RRM
2013 Rustbelt RNA Meeting
RRM
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Poster abstracts
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
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Abstract:
DNA and RNA sequences rich in guanosines (G) can form a four-stranded secondary structure known as a G-quadruplex (GQ), which are found in mammals, fungi and plants and have been extensively studied. They are known to play a role in regulation of gene expression at the transcription and translation level. Both DNA and RNA GQs require monovalent cation (K+) for stabilization of the structures. Effect of divalent metal ions on DNA GQ stability has been studied; however, their effect on RNA GQs remains unknown. We investigated the role of alkaline earth metal cations and a set of divalent transition metal ions on an RNA GQ found within the 5'-untranslated region of matrix metalloproteinase (MT3-MMP) mRNA (M3Q). Our results based upon circular dichroism and RNAse T1 footprinting data reveal that the RNA GQ is destabilized in the presence of the divalent metal cations tested. Transition metal cations are destabilizing the RNA GQ more than alkaline earth cations. However these effects are reversible upon increasing the monovalent cation (K+). We are systematically investigating the characteristic features of the divalent metal ions that are responsible for the destabilization. The results will be valuable in understanding the properties of metal ions that are important to binding and stability of RNA GQs.
References:
1. Neidle, S. and Balasubramanian, S. (2006) Quadruplex Nucleic acid. RSC Biomolecular Science.
2. Mark .J. Morris and Soumitra Basu. (2009) An unusually stable G-quadruplex within the 5' UTR of the MT3 matrix metalloproteinase mRNA represses translation in eukaryotic cells. Biochemistry ACS, 48, 5313-5319.
3. Kathryn Phillips, Zbyszek Dauter, Alastair I. H. Murchie,David M. J. Lilley and Ben Luisi.(1997) The Crystal Structure of a Parallel-stranded Guanine Tetraplex at 0.95AÊ Resolution, J Mol. Biol, 273,171-182.
4. Daisuke Miyoshia, Akihiro Nakaoa, Takeshi Todaa, Naoki Sugimotoa.(2001) Effect of divalent cations on antiparallel G-quartet structure of d(G4T4G4), FEBS letters, 496,128-133.
Keywords: RNA G- quadruplex, divalent metal ions, destabilization
Abstract not available online - please check the printed booklet.
Abstract:
As well established in the central dogma, RNA functions as a key component in the middle of the genetic information transformation chain. With its extensive functional roles and accessible location in the cytoplasm, RNA has been considered as an ideal drug target. Among the variable RNAs, ribosomal RNA (rRNA) has drawn the most attention. Compared to other RNAs, it has a relatively large size with a high level of structural diversity, and its activity directly affects cell survival.
The first FDA-approved, platinum-based anticancer drug, cisplatin, has been widely used in clinical antitumor treatment. Its anticancer activity and mechanism have been throughly studied, and genomic DNA is identified as its primary target; however, recent studies have shown that cisplatin can coordinate and accumulate more rapidly on the RNA than DNA, especially within the ribosome. Without protection of the nucleus or repair mechanisms, RNA has better accessibility as well as sustained adducts compared to DNA. It has been hypothesized that if platinum-based drugs can be modified to target and knock down RNA activity, dose-related side effects can be greatly eliminated. In a previous study, cisplatin was used as a probe to detect the accessible sites in structured RNA. These probing results provided valuable information about the overall
References:
(1) Crick, F. Nature 1970, 227, 561.
(2) Cooper, S. Cell biology international reports 1981, 5, 539.
(3) Lundblad, R. L.; Macdonald, F. Handbook of biochemistry and molecular biology; CRC Press: Boca Raton, 2010.
(4) Trappl, K.; Polacek, N. Metal ions in life sciences 2011, 9, 253.
(5) Franceschi, F. Future Microbiol 2007, 2, 571.
(6) Cohen, S. M.; Lippard, S. J. Progress in nucleic acid research and molecular biology 2001, 67, 93.
(7) Barnes, K. R.; Lippard, S. J. Metal ions in biological systems 2004, 42, 143.
(8) Hostetter, A. A.; Osborn, M. F.; DeRose, V. J. ACS chemical biology 2012, 7, 218.
(9) Rijal, K., Wayne State University, 2011.
Keywords: ribosome, cisplatin analogue, mass spectrometry
Abstract:
The hnRNP H1 protein is a member of the ubiquitously expressed heterogeneous nuclear ribunucleoproteins (hnRNPs) subfamily. hnRNP H1 has three Quasi-RRM domains and Glycine rich repeats. Studies have shown that hnRNP H1 binds poly G segments with high affinity; however, the binding preferences of each sub Q-RRM are unknown1. hnRNP H1, like other members of the hnRNP family functions as a trans regulator of alternative splicing. The mechanism of how hnRNP H1 regulates alternative splicing is poorly understood, however as a step toward gaining a better understanding of hnRNP H1 nucleic acid interactions, the solution structure of the Q-RRM1 domain was determined. The solution structure shows Q-RRM1 consists of four anti parallel beta-strands and three alpha-helices in βαββαβα arrangement. Furthermore we used NMR for determining the amino acid residues interacting with nucleic acid to determine the site of interaction on the Q-RMM1. Additionally isothermal titration calorimetry was utilized for studying biophysical properties of the Q-RRM1 interaction with poly G oligos.
References:
1. Jablonski JA, Caputi M (2009) Role of cellular RNA processing factors in human immunodeficiency virus type 1 mRNA metabolism, replication, and infectivity. J Virol 83: 981–992.
Keywords: hnRNP H, Q-RRM
Abstract:
Pre-mRNA splicing requires proper splice site selection mediated by many factors including snRNPs and serine-arginine rich (SR) splicing factors. Son is the largest known SR splicing factor, and it has several putative functional domains including an RS domain, a glycine rich patch (G-patch) and double stranded RNA binding domain (DSRBD). One-third of Son’s amino acid sequence consists of novel repetitive sequence motifs of unknown function. Son is essential for organization of pre-mRNA processing factors in nuclear speckles and for cell cycle progression. We previously reported an exon array analysis of Son-depleted HeLa cells that revealed changes in 1061 transcripts showing exon inclusion or exclusion, and a total of 2067 splicing events that are potentially regulated by Son. We validated that Son is required for appropriate splice site choice in transcripts for several chromatin-modifying enzymes, including HDAC6, ADA and SETD8. However, the mechanism by which Son maintains accurate splicing is unknown. We are systematically generating model minigene cassettes for molecular and in situ analysis of Son-dependent splicing regulation. We have constructed a HDAC6 minigene reporter that contains the genomic sequence spanning exons 26 through 29. Following Son depletion in HeLa cells transfected with the HDAC6 minigene reporter construct, we observed skipping of exons 27 and 28 on both the reporter and endogenous HDAC6 transcripts. We hypothesize that the C-terminal RS domain of Son is required to maintain proper splicing of HDAC6 transcripts, and we are evaluating siRNA-refractory Son deletion mutants to determine which domains of Son can rescue splicing of HDAC6 minigene transcripts.
Keywords: Spicing, mimigene reporter, Son
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
Liver, the major metabolic organ of the body, undergoes dramatic transitions with regards to structure and function during development. Alternative mRNA splicing (AS) is one of the most prominent mechanisms to generate mRNA complexity, which in turn results in increased proteome diversity. Therefore, we aim to investigate the functional role of AS in mammalian liver development.
To characterize the conserved AS program during liver development, we performed RNA-seq of mouse livers between E18 and P28 timepoints. Among the 150 validated events, 108 (72%) exhibit an increase, and 42 (28%) exhibit a decrease in inclusion of the variably spliced region. Based on this dataset, we performed a temporal analysis of intervening time points, (E16, E18, P0, P2, P7, P14, P28 and P90) and have characterized events that follow prenatal, postnatal and biphasic patterns of splicing. Direct comparison of 118 splicing transitions between mouse and human shows that 57 are evolutionarily conserved during development. Studying these splicing transition networks will be fundamental to understand the regulatory programs that govern development.
We investigated the expression of MBNL1 and CELF1 during liver development and observed that they are strongly down regulated (13 fold and 6 fold respectively) during the first four weeks of liver development. This indicates that changes in splicing factors might play a major role in governing the AS landscape during development. Using knockout and transgenic mice we will further investigate RBPs role in liver development by analyzing the change in splicing patterns due to the absence or presence of these factors. Further studies plan to perform genome-wide iCLIP experiments to determine the direct pre-mRNA targets of these splicing factors.
This study will help us identify conserved mRNA processing transitions in liver development and also establish strong correlations between splicing changes and it’s putative regulators.
Keywords: Alternative Splicing, Liver Development, RNA Binding Proteins
Abstract:
CUG-BP and Elav-like family member 1 (CELF1) is an RNA-binding protein expressed in a variety of tissues, including the embryonic and adult heart, where it is restricted to the cardiomyocytes. CELF1 has been shown to be involved in the regulation of alternative pre-mRNA splicing in the nucleus, as well as transcript degradation and translation control in the cytoplasm. While it is predominantly cytoplasmic in other embryonic tissues, its expression in skeletal and cardiac muscle cells is predominantly nuclear, and this pattern is conserved from frog to mouse. Primary embryonic cardiomyocytes are spontaneously contractile in culture, and this system yields robust knockdown of CELF1 at the transcript and protein levels following siRNA transfection. In a transgenic mouse model in which nuclear CELF activity is repressed in the myocardium as a result of the expression of a dominant-negative CELF protein, profound dilated cardiomyopathy and contractile dysfunction follow. In order to investigate the role of CELF1 in cardiomyocyte contraction, we transfected cultured cells with siRNAs targeting CELF1 and evaluated myofibril structure and function. We observed profound disorganization of myofibrillar structure following siRNA-mediated knockdown of CELF1. By visualizing sarcomeric markers (ACTN2, TTN, and TNNT2) by immunofluorescence, we found a shift in sarcomere appearance from rod-like striations along thick, long fibrils to globular puncta arranged along thin, often web-like, matrices. The consequences of CELF1 abrogation in vivo are currently being investigated. Effects of morpholino-mediated CELF1 knockdown on myofibrillar organization, heart morphology, and cardiac functional parameters are being evaluated in the developing frog embryo by immunofluorescence and optical coherence tomography (OCT).
Keywords: RNA binding protein, myofibrillar structure, cardiac function
Abstract:
To expand our ability to utilize our previously developed comparative analysis of RNA digests (CARD) approach for tRNA analysis,1,2 we must establish reference (baseline) information related to tRNA modifications from beyond the proteobacteria phylum. One phylum of interest is the actinobacteria, which are gram-positive bacteria typically having a high G+C content. To establish a reference set of modifications for us to work with in this phylum, we are presently investigating Streptomyces griseus, which is best known for producing streptomycin. An LC-MS/MS analysis of the modified nucleosides from S. griseus tRNAs yielded 19 unique post-transcriptional modifications. In comparison to Bacillus subtilis, another gram-positive bacterium that is a member of the firmicutes phylum, S. griseus was found to lack mo5U, m5U, s4U, cmnm5U, cmnm5s2U, k2C and Q modified nucleosides. One of the more interesting absences in S. griseus is m5U (rT). This finding was particularly surprising as a BLAST analysis of the TrmA gene from Escherichia coli reveals a gene in S. griseus with high homology. To verify the nucleotide identity at position 54, we used LC-MS/MS to analyze RNase T1 digested S. griseus tRNAs. These experiments reveal that N-54 (in the conserved TPC loop) is either an unmodified uridine or possibly pseudouridine. These findings along with the other differences in S. griseus tRNA modifications will be presented.
References:
1. Siwei Li and Patrick A. Limbach (2012). Method for comparative analysis of ribonucleic acids using isotope labeling and mass spectrometry. Analytical chemistry, 84 8607–8613.
2. Siwei Li and Patrick A. Limbach (2013). Mass spectrometry sequencing of transfer ribonucleic acids by the comparative analysis of RNA digests (CARD) approach. The Analyst, 138 1386–1394.
Keywords: tRNA modification, Streptomyces griseus, mass spectrometry
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
MicroRNA-21 (miR-21) is overexpressed virtually in all human cancers and displays oncogenic activity in a transgenic murine model. Similarly, the p53 tumor suppressor gene is the most frequently mutated gene in human cancer, and its loss or mutation leads to tumor formation in mice. To ascertain the role of miR-21 in the p53 pathway in vivo and to characterize their interaction in tumorigenesis, we intercrossed the miR-21−/− and Trp53−/− mice. We found that Trp53−/−miR-21−/−mice develop tumors at a slightly later age, yet show a similar tumor spectrum and survival curve as Trp53−/− mice. When subjected to genotoxic agents, tissues from Trp53−/−miR-21−/− mice have a higher percentage of apoptotic cells. We extracted mouse embryonic fibroblast cells (MEFs) to examine the impact of miR-21 loss on p53-regulated cellular processes in Trp53−/− cells. Higher cellular apoptosis and senescence were found in Trp53−/−miR-21−/− MEFs than in Trp53−/− MEFs. In addition, loss of miR-21 sensitizes transformed Trp53−/− cells to DNA damage-induced apoptosis through elevation of Pten expression. These data suggest that inhibition of miR-21 would be beneficial in apoptosis-inducing cancer therapies directed against p53-deficient tumors.
Keywords: miR21, Trp53, apoptosis
Abstract:
Human tRNALys3 serves as the primer for reverse transcription in human immunodeficiency virus type-1 (HIV-1). All tRNALys isoacceptors are selectively packaged into budding virions through specific interactions between human lysyl-tRNA synthetase (hLysRS) and the viral Gag protein. tRNALys3 must be released from hLysRS for annealing to the complementary primer binding site (PBS) in the 5’-UTR of the HIV-1 genome in order to initiate reverse transcription. We have shown that this release is facilitated, in part, by the interaction of hLysRS with a tRNA-like element (TLE) in the HIV-1 genome. In HIV-1 NL4-3, which is a clade B subtype, the TLE is located proximal to the PBS and mimics the U-rich anticodon loop of tRNALys. The goal of this project is to establish the conservation of the TLE across various subtypes of HIV-1. We were particularly interested in investigating the HIV-1 MAL isolate, which is a complex recombinant of clades A, D and I. This isolate is representative of clade A and contains a 20-nt insertion near the PBS, which is known to result in an alternative secondary structure fold of the 5’-UTR. We have used fluorescence anisotropy assays to investigate the binding of hLysRS to MAL-derived RNA constructs. The assays reveal moderate to high affinity binding of hLysRS to MAL 123-218 (a 98-mer RNA containing the PBS; Kd = 413±22 nM) and MAL 123-349 (a 229-mer RNA containing the PBS and the dimer initiation signal, DIS; Kd < 50 nM). To locate the sequence elements responsible for the high-affinity binding, truncated genomic RNAs are being designed and tested. An A-rich 23-nt stem-loop RNA previously shown by NMR to mimic the tRNA anticodon structure showed only weak binding to hLysRS (Kd > 5 uM). The results obtained to date suggest differences in the mechanism of tRNALys3 targeting to the PBS and release from hLysRS in different subtypes of HIV-1.
Keywords: HIV-1, TLE, MAL
Abstract not available online - please check the printed booklet.
Abstract:
Long non-coding RNAs (lncRNA) are a novel and abundant category of transcripts which are thought to play critical roles in regulation of diverse aspects of cellular function. Although a role for lncRNAs in several cellular processes has been described, the detailed mechanism behind the observed functions is still very poorly understood. The goal of this project is to advance our understanding of the sequence and structural elements which grant lncRNAs their specific functions. We have chosen a lncRNA as a study model for this project which we have shown to play a crucial role in stress response. In this project, we plan to introduce random mutations into the lncRNA using error-prone PCR. We will construct multiple pools of mutants with theoretical mutation rates which can vary from 50 to 1500 point mutations per molecule. Individual mutant constructs from this library will be cloned into lentiviral vectors and expressed in a pancreatic cell line and screened for resistance to oxidative stress. Mutants which confer a selective advantage against increased oxidative stress will be pooled, enriched and sequenced using high throughput sequencing technologies. The results obtained by this study will provide insights into the functional organization of the sequences within a lncRNA and the way non-coding RNAs can modulate this critical cellular pathway.
Keywords: SELEX, lncRNA, mutagenesis
Abstract:
Aminoacyl-tRNA synthetases are responsible for covalently attaching amino acids to cognate tRNAs though a two-step aminoacylation reaction. Mistakes in this process lead to errors in protein synthesis, and accumulation of such errors can be deleterious to cells. Multiple proofreading mechanisms exist to ensure high fidelity of this key step in protein synthesis. Prolyl-tRNA synthetase (ProRS) can mischarge tRNAPro with alanine and cysteine. The YbaK superfamily of proteins is responsible for hydrolyzing misacylated tRNAPro in all three domains of life. Some bacterial ProRSs possess a cis-editing domain (INS) to hydrolyze Ala-tRNAPro. Cys-tRNAPro and Ala-tRNAPro in some organisms, however, must be cleared by freestanding, trans-editing domains. The bacterial protein ProXp-Ala is responsible for the deacylation of Ala-tRNAPro, yet little is known about their interaction. Mutagenesis studies have identified acceptor stem elements of tRNAPro that are critical for ProXp-Ala activity. Due to this localized specificity, a microhelix that mimics the acceptor stem of tRNAPro was designed and found to be a substrate for ProXp-Ala. No such binding information is available for the protein, however. With the use of Nuclear Magnetic Resonance (NMR) spectroscopy, it is possible to identify specific residues of ProXp-Ala involved in the binding interaction with tRNAPro. After optimizing conditions for multi-dimensional NMR studies of ProXp-Ala, experiments were performed in conjunction with both full-length tRNAPro and the microhelix to map their respective binding sites. Chemical shift perturbations were observed for each, and efforts are underway to assign ProXp-Ala spectra to determine which residues are involved in these interactions. Additionally, Isothermal Titration Calorimetry (ITC) has been performed with both full-length tRNAPro and microhelix to determine the thermodynamics of their interaction, most notably binding affinity and stoichiometry. These experiments and other structural studies will allow me to better characterize the editing complex formed by ProXp-Ala and tRNAPro.
Keywords: tRNA, NMR, ITC
Abstract not available online - please check the printed booklet.
Abstract:
As sessile organisms, plants are often exposed to stress conditions, and have evolved adaptive responses to protect themselves from different types of stress. One of these adaptive systems is that for phosphate (Pi) starvation stress, in which transcription factor PHR1 from the MYB-CC family plays an important regulatory role. In this work, we identified the in vivo targets of PHR1 transcription factor in Arabidopsis genome, using chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) and RNA-seq. PHR1 interacts with 2500 targets, out of which 480 correspond to Pi responsive genes. These targets are enriched in two motifs, P1BS-I and P1BS-II, which are bound by PHR1 using two different modes, as a dimer and as a monomer, respectively. P1BS-I and P1BS-II display different evolutionary constraints, are associated to, in part different P1BS related cis motifs, and their corresponding targets differentially enriched in ontology terms, indicating that PHR1 deploys a dual regulatory logic. We also found that PHR1 targets are enriched in drought induced genes. Physiological, molecular and genetics analysis showed that PHR1 acts as a link between drought responses and Pi homeostasis.
References:
Regla Bustos, Gabriel Castrillo, Francisco Linhares, Maria Isabel Puga, Vicente Rubio, Julian Perez-Perez, Roberto Solano, Antonio Leyva and Javier Paz-Ares (2010) A central regulatory system largely controls transcriptional activation and repression responses to phosphate starvation in Arabidopsis. PLoS Genetics 6, 1-15 e1001102.
Keywords: ChIP-seq, Arabidopsis, PHR1
Abstract:
Cisplatin, or cis-diamminedichlorido platinum (II), is a metal-based anticancer drug widely used to treat different carcinomas. The antitumor activity of cisplatin is believed to be derived from its interactions with DNA, which influences downstream biological functions such as transcription and translation. Besides DNA coordination, cisplatin has been shown to interact with proteins, small sulfur-containing molecules, and RNA. Among different types of RNA inside a cell, ribosomal RNA plays a vital role in protein synthesis. The current work focused on investigating the interactions of ribosomal RNA and cisplatin. Two functionally important RNAs, helix 69 in the large ribosomal subunit and 790 loop in the small subunit, were selected as regions of interest. As an initial approach, small representative RNA constructs were designed for cisplatin reactions. Rates of platination of these constructs were determined by gel analysis. Salt dependence in cisplatin reactivity was observed, consistent with previously published work. Interestingly, naturally occurring nucleotide modifications were found to influence cisplatin reactivity without significant effects on electrostatics. Characterization of the platination sites was done with MALDI mass spectrometry and chemical probing. Consecutive Gs present in the RNA constructs were found to be the primary platination sites. These findings provide interesting insights into metal-RNA interactions found at biologically important regions of ribosomal RNA, which could be valuable targets for future drug design.
Keywords: cisplatin, ribosome , kinetics
Abstract:
The mammalian innate immune system recognizes many viral RNAs through pathogen recognition receptors, including the RNA helicase RIG-I. Function of RIG-I in the innate immune response involves ATP and RIG-I binding to the end of RNA duplexes. However, RIG-I binding to duplex termini does not require ATP, and it is not clear how RNA recognition and ATP utilization are connected. To understand the link between RNA binding and ATP utilization, we examined in vitro how RIG-I couples RNA binding and ATP use for a series of defined model substrates. We find that ATPase activity is only activated by RNA duplexes containing a blunt end. RIG-I binds substrates without duplex termini, but ATPase activity is decreased by more than four orders of magnitude, compared to duplexes with a blunt end. Our observations further show that RIG-I oligomerizes on duplex RNAs, but only the terminal protomer bound to a blunt end hydrolyzes ATP. Collectively, our data indicate that RIG-I utilizes the ATP to proofread substrates for blunt duplex ends.
Keywords: RIG-I
Abstract:
Enzymes that are site-specifically labeled using fluorescent dyes or biotin are particularly useful for bulk and single molecule biochemical investigations. However, the methods for site-specific labeling of enzymes use complex molecular biology techniques which can often result in enzymes with significant loss of function. Here we describe a straightforward and mild technique for N-terminal labeling and conjugation of a protein using a combination of native chemical ligation (NCL) and copper-mediated azide-alkyne cycloadditon ('click chemistry'). For the NCL reaction an azido lysine thioester molecule (azido-NCL-adapter) was synthesized starting from commercial Boc-Lysine in three simple steps. Initially, the condition for NCL using the azido-NCL-adapter was optimized with a test peptide that included an N-terminal cysteine. Subsequently, the peptide could be used in click-chemistry reactions that furnished labeled or conjugated peptide. With these optimized conditions we advanced to the lariat debranching enzyme (Dbr1p) that was expressed with an N-terminal cysteine. The Dbr1p cysteine reacted under NCL conditions with the azido-NCL-adapter to generate an azido-modified Dbr1p. This azido labeled enzyme could be conjugated either to a fluorescent dye (Alexa555) or to a biotinylated DNA sequence using 'click chemistry'. The biotin-DNA tether on the N-terminus of Dbr1p can serve to immobilize the enzyme on a surface for single molecule experiments. The Dbr1p specifically cleaves the 2'-5' phosphodiester bond of lariat RNA. The modified enzyme - labeled or conjugated - showed retention of the debranching activity in cleaving 2'-5' phosphodiester linkage of a backbone branched RNA substrate. Such site-specifically modified debranching enzymes will be used for single molecule experiments in the future. As well, we envision our straightforward method for site-specific labeling or conjugation will find more general use for the investigation of other proteins.
Keywords: Native chemical ligation, click-chemistry, site specific labeling
Abstract:
Ribosome biogenesis is a complex, multistep process that involves processing and folding of rRNA and binding of ribosomal proteins. Previous research has shown that many different classes of trans-acting proteins are also required for assembly, with both enzymatic and structural functions. However, the precise functions of most of these assembly factors are not known. To begin to explore roles of assembly factors in ribosome biogenesis, we investigated their functional relationships with ribosomal proteins. Although a significant amount of information exists about roles of both ribosomal proteins and assembly factors in ribosome biogenesis, not much is known about the interplay between the two types of proteins. Previous research has shown order of assembly of ribosomal proteins into pre-ribosomes, as well as with which assembly factors they associate. However, most of these studies started with specific ribosomal proteins and then asked: with which assembly factors do they interact, and which assembly factors depend on them to associate with pre-ribosomes?
Specifically, we asked which ribosomal proteins depend upon the assembly factors Nog1, Nog2, Dbp10, and Spb4 to assemble. These assembly factors were selected because they are thought to have similar functions (as GTPases or ATPases). Ribosomal proteins were tagged in strains conditional for expression of these assembly factors, and association of ribosomal proteins with pre-ribosomes was explored upon depletion of each of these factors. This work is intended to complement the work that has already been done by providing a more complete picture of how these ribosomal proteins fit into the established recruitment pathway for assembly factors. Because there is much more structural information about position and conformation of ribosomal proteins on the pre-ribosome, this work will hopefully elucidate similar information about the assembly factors. This approach has benefitted us because we have reevaluated out the previously established assembly hierarchy. This work has also lead us to believe that it is not simply RNA processing that allows these ribosomal proteins to stably associate with the pre-ribosome, but the presence of these assembly factors is required prior to the processing of RNA.
Keywords: ribosome assembly, ribosomal proteins, ribosome assembly factors
Abstract not available online - please check the printed booklet.
Abstract:
The lack of new antibiotics is a growing concern due to an increase in bacterial resistance across the different classes of known drugs. One limitation of the current antibiotics is their overlapping binding sites on the target ribosome; therefore, novel sites are essential for the success of new antibiotics. Helix 69 (H69) is a 19-nucleotide region of the 50S ribosomal subunit. Although the exact function of H69 is currently unknown, it has been implicated to have important roles in ribosomal processes such as subunit association, ribosomal recycling, and tRNA selection. Conserved across phylogeny, bacterial H69 also contains three pseudouridine residues. To determine the drug potential and functional applications of the H69 motif, phage display was used to reveal a heptameric peptide sequence that binds to H69 with moderate (low micromolar) affinity and selectivity. Although this technique is useful in identifying moderate binders, tight-binding sequences may not be found due to the target's implicated role in various translational processes. To surpass this limitation, chemical modifications or mutations of the parent sequence were made by using solid-phase peptide synthesis. Relative dissociation constants for the RNA-peptide complexes were determined by using electrospray ionization mass spectrometry. Modified peptides with greater binding affinity than the parent were further characterized by using a variety of biophysical techniques. A visual assay was developed to further screen peptide libraries to choose novel ligands that can be further characterized and tested for binding and selectivity to H69 RNA. The work described supports that natural, specific ligands can be identified to target oligonucleotide targets and improved binding can be achieved with the use of chemical modifications. Furthermore, H69 functionality can be elucidated by using these peptide ligands combined with biological and biophysical techniques making this experimentation applicable to other oligonucleotide constructs implicated in essential biological processes.
Keywords: Ribosome, Helix 69, Peptide Ligands
Abstract:
Aminoacyl-tRNA synthetases (aaRS) are enzymes primarily responsible for charging tRNAs with their cognate amino acid, however, most have alternative functions. A subset of higher eukaryotic aaRSs are associated with a multi-synthetase complex (MSC). Human lysyl-tRNA synthetase (LysRS) is normally a core member of the MSC, but is packaged into HIV-1 particles through interactions with the Gag polyprotein. This interaction brings host cell tRNALys3, which serves as the replication primer, into virions. Disruption of this interaction, and subsequent packaging, inhibits infectivity of HIV-1, underscoring the importance of this protein in the viral lifecycle. The mechanism by which LysRS associates with HIV-1 components is unclear. During HIV-1 assembly, Gag forms a series of transient assembly intermediates, some of which contain an ATP-binding protein, ABCE1. These complexes were isolated by sucrose gradient with sedimentation coefficients ranging from 10S to 500S, and immunoprecipitated to extract ABCE1 assembly complexes. We have shown that both LysRS and tRNALys3 are present in the 80S, 150S and 500S fractions. Reverse transcriptase assays are underway using samples containing tRNALys3, to determine if the tRNA is annealed to genomic RNA prior to viral budding. To further examine LysRS trafficking, immunofluorescence is being used to co-localize endogenous LysRS and GFP-reporter HIV-1 in HEK293T and CD4+ HuT/CCR5 T-cells.
Keywords: LysRS, HIV, tRNA
Abstract not available online - please check the printed booklet.
Abstract:
A Small protein that interacts with EF-Tu, GluRS2 and small molecules
Shirin Fatma, Keng-Ming Chang and Tamara L. Hendrickson*
Department of Chemistry, Wayne State University, 5101 Cass Avenue, Detroit, MI 48202. USA.shirin@chem.wayne.edu
Maintaining the fidelity of protein biosynthesis is crucial to all life but offers unique challenges in organisms missing one or more of the aminoacyl-tRNA synthetases (aaRSs). H. pylori, an ɛ-proteobacterium, is missing both glutaminyl-tRNA synthetase (GlnRS) and asparaginyl-tRNA synthetase (AsnRS) and synthesizes both Gln-tRNAGln and Asn-tRNAAsn by an indirect aminoacylation pathway. To generate Asn-tRNAAsn, tRNAAsn is mischarged by a non-discriminating aspartyl-tRNA synthetase (ND-AspRS). Next, Asp-tRNAAsn is converted to Asn-tRNAAsn by an amidotransferase (AdT). However, for production of Gln-tRNAGln, H. pylori have two functional copies of gltX, the gene encoding for glutamyl-tRNA synthetase. GluRS2 specifically generates Glu-tRNAGln, which is subsequently converted to Gln-tRNAGln by the same AdT. Partly because of these misacylation pathways, protein biosynthesis includes different proofreading checkpoints (e.g. within the aaRSs, EF-Tu, and the ribosome).
We are examining proteins of unknown functions that were identified by yeast two-hybrid (Y2H) as potential new players in tRNA aminoacylation (both direct and indirect) and fidelity. These efforts have identified Hp0495 as a possible participant. Hp0495 binds ATP, glutamate, and either tRNAGlu1 or tRNAGln. It also forms a complex with EF-Tu and a tRNAGln-dependent complex with GluRS2. These results suggest that Hp0495 plays a role in preventing Glu-tRNAGln complex formation with EF-Tu or in delivering this misacylated tRNA to AdT. The possible consequences of these ribonucleoprotein complexes on tRNA aminoacylation, transamidation, and translational fidelity will be discussed. Existence of these proteins of unknown function and their interactions with components of the translation machinery clearly demonstrates that the classical boundaries of the field of tRNA aminoacylation need to be expanded.
Keywords: EF-Tu
Abstract:
RNA Polymerase II (RNAPII) is the enzyme responsible for the transcription of messenger RNA (mRNA) and many small non-coding RNAs. While much is known about RNAPII transcription initiation and its role in expression regulation, eukaryotic termination mechanisms are still active areas of investigation. There are two described RNAPII termination mechanisms: most mRNAs are terminated by the polyadenylation dependent cleavage and polyadenylation complex and the Rat1 exonuclease while most noncoding RNAs and unstable transcripts are terminated by the Nrd1 complex and nuclear exosome. The nuclear exosome has an important role in RNA surveillance, processing, and degradation of a wide range of RNAs. The nuclear exosome is specifically required for 3’end trimming of small noncoding RNAs, and deletion of RRP6 causes accumulation of long, untrimmed transcripts (read-through transcription) as well as altered RNA expression levels. Disruptions in proteins that control RNAPII phosphorylation, such as Rtr1 and Ssu72, also cause read-through transcription and altered expression to different extents. Using RNA-sequencing data of total RNA isolated from S. cerevisiae, we are developing methods to identify read-through transcripts and altered expression levels genome-wide in rrp6Δ cells. This will allow us to identify novel targets of the nuclear exosome and gain a better understanding of termination pathway usage. Previous studies of RRP6 targets using titling arrays had limited dynamic range compared to RNA-sequencing, and we are able to detect a much larger difference between short, processed small noncoding RNAs which are extremely abundant and the less abundant read-through transcripts that accumulate when RRP6 is deleted. Read-through transcripts, even in exosome mutants, are a small fraction of the transcripts produced, and the depth of sequencing at these abundant snRNAs make them excellent targets for analysis. This study will allow us to optimize our analysis pipeline that can then be used to analyze the effects of disrupting other termination factors and proteins involved in RNAPII phosphorylation.
Keywords: RNA sequencing, exosome, 3 end processing
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
The eukaryotic translation initiation factor 4F (eIF4F) contains the large scaffold protein eIF4G, the cap-binding protein eIF4E, and the loosely associated DEAD-box RNA helicase eIF4A. In Saccharomyces cerevisiae, another DEAD-box RNA helicase Ded1p also interacts with eIF4G. To understand how the Ded1p-eIF4F interaction impacts the molecular function the proteins, we examined the consequences of this interaction on the biochemical activities of the eIF4F components and of Ded1p, using reconstituted complexes. We show that Ded1p allows eIF4G/eIF4E to associate with short RNAs that eIF4G/eIF4E alone cannot bind, and that Ded1p and eIF4G/eIF4E facilitate each other's binding to longer RNAs. eIF4G/eIF4E enhances the RNA-stimulated ATP hydrolysis by Ded1p, and establishes the most stable interaction with Ded1p when the helicase is bound to RNA and ATP. At the same time, eIF4G/eIF4E inhibits RNA unwinding by Ded1p by interfering with oligomerization of Ded1p, which is needed for optimal unwinding activity. However, addition of eIF4A to the eIF4G/eIF4E complex stimulates unwinding activity by Ded1p, even though eIF4A contributes only negligible unwinding activity on its own. Nonetheless, this stimulation requires ATP hydrolysis by eIF4A. We also show that Ded1p enhances the binding of eIF4E to the RNA 5’-cap, but only in the presence of eIF4G. Collectively, our results show that the Ded1p-eIF4F interaction impacts multiple biochemical properties of Ded1p and eIF4F, and that this modulation requires two functional DEAD-box helicases. This synergism suggests that both eIF4A and Ded1p can work simultaneously in conjunction with eIF4G and eIF4A.
Keywords: DEAD-box helicase, eIF4F, Ded1p
Abstract:
In almost all bacteria and archaea, translation of the isoleucine codon AUA requires a modified C (lysidine or agmatidine) at the wobble position of tRNA Ile to base pair specifically with the A of the AUA codon but not with the G of AUG. Recently, a Bacillus subtilis strain was isolated in which the gene encoding tRNAIle-lysidine synthetase was deleted. Consequently, the wobble base C34 of tRNA Ile remains unmodified and cells depend on a mutant tRNA derived from tRNA Ile, in which G34 at the wobble position has been changed to U34. Here, we show (i) that unlike U34 in the wobble position of most other tRNAs, U34 of the mutant tRNA is not modified, and (ii) that the mutant tRNA binds preferentially to the AUA codon on B. subtilis ribosomes but only weakly to AUG. These in vitro data are consistent with the finding that the suppressor strain displays only a low level of misreading AUG codons in vivo and grows at a rate comparable to that of the wild type strain.
Keywords: tRNA, Mass Spectrometry, RNA modification
Abstract:
Adenosine Deaminases Acting on RNA (ADARs) have been studied in many animal phyla, where they have been shown to deaminate specific adenosines into inosines in duplex mRNA regions. In Drosophila, two isoform classes are encoded, designated full-length (contains the editase domain) and truncated (lacks this domain). Much is known about the full-length isoform, which plays a major role in regulating functions of voltage-gated ion channel proteins in the adult brain. In contrast, almost nothing is known about the functional significance of the truncated isoform. In situ hybridization shows that both isoform mRNA classes are maternally derived and transcripts for both localize primarily to the developing central nervous system. Quantitative RT-PCR shows that about 35% of all dADAR mRNA transcripts belong to the truncated class in embryos. 3’-RACE results show that abundance of the truncated isoform class is developmentally regulated, with a longer transcript appearing after the mid-blastula transition. 3’-UTR sequences for the truncated isoform have been determined from diverse Drosophila species and important regulatory regions including stop codons have been mapped. Western analysis shows that both mRNA isoform classes are translated into protein during embryonic development, as full-length variant levels gradually diminish. The truncated protein isoform is present in every Drosophila species studied, extending over a period spanning about 40 x 106 years, implying a conserved function. Previous work has shown that a dADAR protein isoform binds to the evolutionarily conserved rnp-4f pre-mRNA stem-loop located in the 5’-UTR to regulate splicing, while no RNA editing was observed, suggesting the hypothesis that it is the non-catalytic truncated isoform which regulates splicing. To test this hypothesis, we have utilized RNAi technology, the results of which support the hypothesis. These results demonstrate a novel, non-catalytic function for the truncated dADAR protein isoform in Drosophila embryonic development, which is very likely evolutionarily conserved.
Keywords: dADAR gene, RNAi knockdown, intron splicing regulation
Abstract not available online - please check the printed booklet.
Abstract:
Ptbp2 is the brain and testis expressed paralog of polypyrimidine tract binding protein PTB. We have previously shown that Ptbp2 is essential for postnatal survival in mice, and functions predominantly as a silencer of alternative exon splicing in the embryonic brain to repress the expression of developmentally regulated ‘adult-enriched’ exons(1). To determine if tissue-restricted RNA binding proteins have the same or tissue-specific functions in RNA regulation in different biologic contexts we investigated the role of Ptbp2 in mouse testis. We generated Ptbp2 HITS-CLIP libraries from whole testis and performed comparative analyses with HITS-CLIP data generated from embryonic brain. This analysis identified RNA targets in which Ptbp2 binding is identical in brain and testis, and RNA targets where Ptbp2 exhibits tissue-specific differences in RNA binding suggestive of testis-specific and splicing-independent roles for Ptbp2 in RNA regulation. Using mice bearing a conditional ‘floxed’ allele of Ptbp2 (generously provided by Qin Li and Doug Black, UCLA) and the germ cell-specific Stra8-iCre driver(2), we have generated mice with Ptbp2-null germ cells. Loss of Ptbp2 results in spermatogenic arrest following the production of haploid spermatids. Strikingly, chains of haploid spermatids (normally held together by intercellular bridges) coalesce to form giant multinucleate cells in Ptbp2-null testis. These observations indicate that RNA regulatory functions performed by Ptbp2 in germ cells are essential for mammalian germ cell development. To explore the functions of Ptbp2 in specific stages of germ cell development, we have developed a new germ cell purification strategy using a dual fluorescence RFP/GFP transgene(3) and flow cytometry to isolate wild type and Ptbp2-null germ cells in each of the major stages of spermatogenesis. This approach will allow us to investigate the roles of Ptbp2 as germ cells progress through different stages of germ cell development, thus providing valuable insights into how a single RNA binding protein regulates its RNA targets in different cell types in an in vivo model of mammalian cell development.
References:
1)Licatalosi et al, 2012 Genes Dev 26, 1626-1642.
2)Sadate-Ngatchou et al, 2008 Genesis 46 738-742
3)De Gasperi et al, 2008 Genesis 46 308-317.
Keywords: spermatogenesis, CLIP, splicing
Abstract:
RanGAP is the activating protein for the small GTPase Ran, and is involved in nucleocytoplasmic transport and mitotic cell division across kingdoms. Arabidopsis has two RanGAP genes, RANGAP1 and RANGAP2, which have partially redundant function. We recently discovered that a RANGAP1 RANGAP2 double null mutant is female gametophyte lethal, arresting after the first mitotic division following meiosis. During open mitosis, RANGAP1 is targeted by a plant-specific domain to sites important for mitosis/cytokinesis, and depletion of RANGAP1 transcripts leads to cell wall placement defects. A mutant combining a RANGAP1 null allele with severely decreased RANGAP2 expression has severe defects in sporophyte development, but no apparent defect in protein nuclear import. We hypothesize that these data reveal novel, plant-specific roles for RanGAP. To determine whether RanGAP’s canonical GTPase activation (GAP) activity and/or its unique mitotic localization are required for wildtype RanGAP function, we have generated point mutations and deletions in RANGAP1 that disrupt its GAP activity, subcellular targeting, or both. We then determine whether these mutant proteins rescue female gametohpyte lethality and sporophyte development. Additionally, we investigate the role of plant RanGAP in nucleocytoplasmic trafficking of RNAs and large protein complexes.
Keywords: RanGAP, Ran, Arabidopsis
Abstract:
The highly conserved and essential DEAD-box helicase Ded1p is critical for translation initiation in Saccharomyces cerevisiae. To understand how molecular function of Ded1p corresponds to its role in translation initiation we used a temperature-sensitive Ded1p mutation that causes slow growth at elevated temperatures. This mutation lowers affinity of Ded1p to RNA and alters the kinetic response of translation initiation to mild heat shock (30oC to 37oC). RNA-seq of the translated mRNA pool shortly after heat shock shows that the RNA binding defect in Ded1p impacts translation initiation for most mRNAs. Translation initiation is decreased for roughly 1/3 of the affected mRNAs, and increased for the remaining 2/3. The largest decrease is seen for mRNAs encoding proteins involved in mRNA metabolism. We further show that Ded1p binds to a large cross-section of mRNAs, consistent with functional effects on a broad range of mRNAs. Ded1p binds mRNAs at multiple sites that do not show obvious sequence or structure signatures. However, the pattern of Ded1p binding differs between more and less affected mRNAs. Collectively, our data indicate that Ded1p is a general translation initiation factor that affects translation initiation of virtually all RNAs. However, the magnitude of the effects corresponds to the binding pattern of Ded1p to a given mRNA.
Keywords: RNA helicase, translation initiation, RNA binding
Abstract:
While the long non-coding RNAs constitute a large portion of the mammalian transcriptome, the mechanistic aspects of their biological functions have remained mostly elusive. To gain insight into the relationship between the function of a long non-coding RNA and the complement of cellular factors with which it interacts, we have developed an in vivo pull down approach. This method is based on the use of biotinylated antisense oligos (ASOs) to pull down a targeted RNA and its associated proteins from cellular extracts. To ensure that the observed RNA-protein interactions reflect those occurring in vivo, cells are crosslinked prior to preparation of extracts. In order to distinguish the bona-fide “interactome” proteins that copurify with the target RNA from those that are present in the pull down fraction due to possible off-target interactions of ASOs, at least six different ASOs will be used. These will include four non-targeting ASOs and at least two ASOs that are complementary to different sequences in the target RNA and will be individually used in six parallel pull down reactions. The co-purified proteins will be identified by mass spectrometry and those that are co-purified with both targeting ASOs but are not found in pull down fractions obtained with non-targeting ASOs, will be identified as part of the interactome of the targeted RNA. We have successfully employed this method on BORG lncRNA and have identified ~ 20 proteins which specifically and reproducibly co-purified with BORG. We have confirmed the mass spectrometry results by western blot, and importantly, have been able to validate the interactions captured by lncRNA-PICASO by reciprocal immunoprecipitation of the identified proteins followed by RT-PCR for BORG (RNA IP). This method permits the purification of endogenous lncRNAs without a need for addition of sequence elements such as MS2 loops, which may interfere with biogenesis or function of lncRNAs. Our results suggest that lncRNA-PICASO is a robust method for defining the interactome of both lncRNAs and mRNAs. Further, the described method is sensitive enough for purification of relatively low abundance transcripts, which makes it applicable to a wide range of cellular long non-coding transcripts.
Keywords: long non-coding RNA, interactome
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
Usher syndrome (Usher) is the leading genetic cause of combined deafness and blindness. Type 1 Usher (Usher 1) is the most severe form of the disease and is characterized by hearing impairment and vestibular dysfunction from birth, and the development of retinitis pigmentosa (RP) in early adolescence. A 216G>A (216A) mutation in USH1C accounts for all Usher 1 cases in Acadian populations of the United States and Canada. The USH1C gene codes for the protein harmonin which is essential for development of hair cells in the ear and survival of retinal cells. The 216G>A mutation creates a cryptic splice site that is used preferentially over the authentic splice site. Use of the cryptic site gives rise to a frame-shift that results in the production of a truncated, non-functional protein.
We developed an antisense oligonucleotide, called ASO-Ush, which binds to the 216A mutation in USH1C RNA and blocks it from being recognized by the cellular splicing machinery and splicing is re-estalished at the authentic splice site. In this way, the deleterious effects of the mutation are subverted and harmonin expression restored. The chemistry of ASO-USH makes the molecule very stable and easily deliverable to different cell types in the body. The specificity of the molecule eliminates harmful side-effects that typically arise from off-target effects of drugs.
ASO-USH was tested for its ability to block the 216A mutation and restore harmonin expression in a mouse model of Usher syndrome. These mice were engineered to have the identical Ush1c.216G>A mutation (216AA) as humans. These Usher mice are deaf, exhibit circling behavior indicative of severe vestibular dysfunction and have retinal dysfunction by 1 month of age and begin to lose photoreceptors between 6 and 12 months of age. We found that mice treated with a single dose of ASO-USH shortly after birth had normal vestibular function and could hear for up to six months of age. The treated mice also had a modest improvement in visual function at one month of age. Our results demonstrate that ASO-USH can effectively correct an Ush1c mutation and suggest the therapeutic potential of this class of molecule in Usher syndrome and other diseases of the eye and ear.
Keywords: antisense oligonucleotide, splicing, Usher syndrome
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
Helix 69 (H69), a 19-nucleotide segment located within domain IV of the 23S ribosomal RNA, is a key component of the intersubunit bridge B2a in ribosomes. The sequence and secondary structure of H69 are highly conserved in each domain of life and throughout the phylogeny. An additional feature of conservation of H69 is a cluster of pseudouridine (Ψ) modifications within the loop region. NMR studies on oligonucleotides with and without Ψs have shown that these modifications reshape and influence the dynamic behavior of the H69 loop region. This structure modulation by Ψ modification is proposed to be correlated with a growth advantage of wild-type strains and conservation of Ψs. Meanwhile, it has been reported that H69 plays important structural and functional roles by displaying conformational adaptability, involving association with different factors such as Mg2+. The folding of H69 is also susceptible to environmental pH changes. NMR studies on H69 oligonucleotides with and without Ψ modifications suggest that the pseudouridylated H69 is more sensitive to environmental pH change than the unmodified construct, and reveal at least two Mg2+ binding sites. Thus, incorporation of environmental changes in the NMR studies enriches our understanding of the roles of Ψ in H69 structure and conformational behavior, and extends our knowledge towards the structures in physiological conditions. The results of these model studies are useful in research and development of antibiotic candidates that target H69.
References:
Jiang, J., Sakakibara, Y., Chow, C.S. (2013) Israel Journal of Chemistry 53: 379-390.
Goddard, N.L., Bonnet, G., Krichevsky, O. and Libchaber, A. (2000) Physical review letters 85: 2400-2403.
Sakakibara, Y., Abeysirigunawardena, S.C., Duc, A-C.E., Dremann, D.N., Chow, C.S. (2012) Angewandte Chemie International Edition 51: 12095-12098.
Blaha, G., Stanley, R.E., Steitz, T.A. (2009) Science 325: 966-970.
Klinge, S., Voigts-Hoffmann, F., Leibundgut, M., Arpagaus, S., Ban, N. (2011) Science 334: 941.
Keywords: pseudouridylation, pH, magnesium
Abstract:
Cardiac dysfunction is the second leading cause of death in myotonic dystrophy type 1 (DM1). A screen of more than 500 miRNAs in a DM1 mouse model identified 54 miRNAs that were differentially expressed in heart. More than 80% exhibited down regulation towards the embryonic expression pattern and showed a DM1-specific response. Twenty of the 22 miRNAs tested were also significantly down regulated in DM1 human heart tissue. We found that a large proportion of these miRNA are Mef2 transcriptional targets. MEF2A and MEF2C protein expression is significantly reduced in both human DM1 heart samples and the mouse model. Exogenous Mef2C expression restores normal levels of Mef2 target miRNAs and mRNAs in a DM1 cardiac cell culture model. These results indicate that mis-regulation of cardiac Mef2 circuitry is causal to the reprogramming of gene expression in DM1.
Keywords: Alternative splicing, microRNA, Myotonic Dystrophy
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
The hepatitis delta virus (HDV) is a small, satellite RNA virus of the hepatitis B virus. HDV infection results in severe acute hepatitis with a high risk of chronic liver disease progressing to cirrhosis. Replication of HDV requires the activity of the HDV ribozyme (HDVrz), a small, catalytic RNA motif that undergoes a self-cleavage reaction to produce viral unit-length genomes. Understanding how the HDVrz catalyzes self-cleavage is important for understanding mechanisms of viral pathogenesis and provides important insights into biological catalysis. We have developed methods for measuring the relative catalytic activity of multiple alternative substrates in a single reaction by quantifying the ratios of substrate concentrations as a function of reaction progress using an internal competition assay. Here, we apply this multiple alternative substrate kinetic approach to the HDVrz, in order to provide a systematic analysis of proposed interactions between the substrate and HDVrz. Previous molecular mechanics simulations of the HDVrz reaction indicated the potential for formation of a Watson-Crick base pair between the catalytic RNA and its substrate that is necessary in positioning the nucleophile for catalysis. The modeling implicates that A79 in the J 4/2 region of the ribozyme forms a canonical Watson-Crick base pairing interaction with the U(-1) on the substrate. To test this hypothesis we created mutant enzymes and substrates with all four nucleotide possibilities at each position. Disruption of the base pair between A79:U(-1) should decrease catalytic activity, while compensatory mutations that restore base paring would be expected to rescue activity. Additionally, as the nucleobase identity of the (-1) position on the substrate strongly influences HDVrz catalytic activity, we have created substrate mutants containing nucleotide analog substitutions to provide further insight into the specific interactions of the (-1) position with the ribozyme.
Keywords: catalytic RNA, HDV ribozyme
Abstract not available online - please check the printed booklet.
Abstract:
Regulation of Endothelin-1, the most potent vasoconstrictor, has a pathophysiological importance in cardiovascular diseases (hypertension, coronary heart diseases, congestive heart failure, and atherosclerosis), pulmonary diseases (asthma and hypertension), central nervous system diseases (Hirschprung’s disease, stroke and subarachnoid hemorrhage), and eye diseases (diabetic retinopathy and glaucoma). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a glycolytic enzyme, can bind to the adenine-uridine rich elements (AREs) in the 3’untranslated region (3’UTR) of Endothelin-1 (ET1) mRNA, destabilize its mRNA, and leads to decreased ET1 protein levels. Pathophysiological implications of ET1 dysregulation have been treated clinically by targeting ET1 receptors or ET1 processing proteases. GAPDH-mediated regulation of ET1 mRNA stability offers an alternative therapeutic intervention. However, the molecular mechanism by which GAPDH binds to and destabilizes ET1 mRNA is unknown. GAPDH does not contain sequence homology to RNA recognition domain like other reported AdenineUridine binding proteins (AUBPs), however our preliminary studies suggest that GAPDH may contain a classical RRM like structural motif that could be involved in RNA binding.
We aim to determine the specific interactions between GAPDH and ET1 mRNA, by using mutagenesis, RNA electrophoretic mobility shift assay (REMSA), fluorescence anisotropy, molecular modeling, and xray crystallography.
We will use this structural information to guide structure based drug design of small molecules aimed at destabilizing ET1 mRNA and controlling cellular ET1 protein levels to treat cardiovascular diseases.
References:
J. Cardiovasc. Res. (2007) 76, 8–18
The FASEB Journal (2011) 25, 16–28
Bioorg Med Chem Lett. (2000) 10, 2037-9
J Cardiovasc Pharmacol (2000) 36, S36-9
Mol. Cell. Biol. (2008) 28, 7139–7155
Biochem. J. (2005) 387, 763-772
J Neurochem. (2003) 85:1228
Cell Signal. (2011) 2392:317-323
Science (1994) 265, 615-621
J. Biol. Chem. (2004) 279(10), 8655–67
J. Biol. Chem. (1995) 270, 2755–2763
Nat. Chem. Biol. (2008) 4, 700-707
J. Biol. Chem. (2005) 280, 22406-22417
Keywords: Endothelin-1, RNA, vasoconstriction, post-transcriptional regulation, heart diseases, RRM motifs, adenine-uridine rich elements (AREs)
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
Ribonucleotide reductase (RR) converts RNA nucleotides to DNA nucleotides, and is an attractive and well-established chemotherapy target, but present drugs are largely nucleotide inhibitors which target the enzyme’s active site (1). The nature of these compounds predisposes them to causing deleterious off-target effects which have no therapeutic value (2, 3). A demand therefore exists for RR inhibitors which are not nucleotide analogs. Our group seeks to identify such compounds by a combination of in vivo and in vitro drug screens for compounds which can disrupt pools of dNTPs in vivo, and which alter the relative second-order rate constants for RR’s four substrates (A, G, C, UDP) in vitro. This disruption of dNTP pools will interfere with DNA replication and repair without disrupting other essential processes, slowing tumor growth and potentiating radiation therapy (4, 5). The normal balanced pool of dNDPs is generated via an elegant crosstalk system between the active site of the large subunit and an allosteric site called the specificity site, where dNTP effectors (ATP/dATP, dGTP, dTTP) bind and modulate RR’s preferences among its substrates (6). Disrupting this allosteric transfer of information is vital for the desired perturbation of dNTP pools. We will observe relative rates of formation for each of the four dNDP products in vitro using borate chromatography and HPLC. Because each substrate acts as a competitive inhibitor for the other three, determining the relative rates of formation at known substrate concentrations is sufficient for the determination of relative second-order rate constants (7). We will use this assay to interrogate the effects of newly screened compounds known to inhibit RR in human cancer cell lines. These data will provide insight into the mechanisms of action for the screened compounds, which will lead to iterative rational design of progressively more effective chemotherapeutic agents.
References:
1. Wijerathna, S.R.; Ahmad, M.F.; Xu, H.; Fairman, J.W.; Zhang, A.; Kaushal, P.S.; Wan, Q.; Kiser, J.; Dealwis, C.G. Pharmaceuticals 2011, 4, 1328-1354.
2. Burstein, Harold J. Journal of Clinical Oncology 18.3 (2000): 693-693.
3. Galmarini, Carlos M., John R. Mackey, and Charles Dumontet. The lancet oncology 3.7 (2002): 415-424.
4. Shewach, Donna S., and Theodore S. Lawrence. Investigational new drugs 14.3 (1996): 257-263.
5. Oliver, F. J., M. K. L. Collins, and A. Lopez-Rivas. Experientia 52.10-11 (1996): 995-1000.
6. Xu, Hai, et al. Proceedings of the National Academy of Sciences of the United States of America 103.11 (2006): 4022-4027.
7. Cornish-Bowden, Athel. Journal of theoretical biology 108.3 (1984): 451-457.
Keywords: Enzymology, Drug Design
Abstract:
Ribosome assembly is a complex biological process in which ribosomal RNA (rRNA) is transcribed in precursor form, processed, modified, and bound by ribosomal proteins to generate the mature ribosomal subunits. Many trans-acting proteins (~200) called ‘assembly factors’ are essential for ribosome assembly in yeast and higher organisms. To better understand how these assembly factors aid ribosome assembly, our lab has focused on a subset of evolutionarily conserved assembly factors called the ‘A3-factors’ (Nop7, Erb1, Ytm1, Rlp7, Nop15, Cic1, Nop12, Ebp2, Brx1, Has1, Pwp1, Drs1), required for the exonucleolytic processing of the 27SA3 precursor rRNA (pre-rRNA)(2,3).
Our research focuses on three A3 factors Nop7, Erb1, and Ytm1, which form a heterotrimeric subcomplex. Analyses of amino acid sequences of these proteins suggest that they lack enzymatic activity, and are likely to serve as structural proteins. Recent research indicates that the Nop7-subcomplex facilitates pre-rRNA folding by enabling bridging of domains I and III of 25S rRNA via protein-protein and protein-rRNA interactions during formation of tertiary structure of the 60S subunit(1,3). Our goal is to understand the role of Nop7 subcomplex proteins in establishing the structure of pre-ribosomes. We discuss a systematic approach to generate specific protein-protein and protein-RNA interaction mutants and describe effects of the mutations on 60S subunit assembly.
References:
1.Granneman, S., Petfalski, E., Tollervey, D. (2011) A cluster of ribosome synthesis factors regulate pre-rRNA folding and 5.8S rRNA maturation by the Rat1 exonuclease. EMBO J 30: 4006-4019.
2. Sahasranaman, A., Dembowski, J., Strahler, J., Andrews, P., Maddock, J., Woolford, J.L., Jr. (2011) Assembly of Saccharomyces cerevisiae 60S ribosomal subunits: role of factors required for 27SA3 pre-rRNA processing. EMBO J. 30: 4020-4032.
3. Tang, L., Sahasranaman, A., Jakoljevic, J., Schleifman, E., Woolford, J.L. Jr. (2008) Interactions among Ytm1, Erb1, and Nop7 required for the assembly of Nop7-subcomplex in yeast preribosomes. Mol. Biol. Cell. 19: 2844-2856.
Keywords: Ribosome Biogenesis, RNA folding
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
The function and core structure of ribosomes are conserved in all kingdoms of life. Consistent with the increased complexity of their function, eukaryotic ribosomes are larger than their bacterial counterparts. Eukaryote-specific extensions of ribosomal proteins (r-proteins) and expansion segments of rRNAs account for this size difference. These additions are thought to have roles in translation regulation and may increase the stability of ribosomes through extra protein-protein and protein-rRNA interactions. However, specific roles have been assigned to only a few of extensions. Interestingly, some regions within extensions are structurally disordered and transition to an ordered state upon binding their ligand, but the significance of this structural feature in ribosome assembly is yet to be determined. Our goal is to investigate whether eukaryotic extensions have roles in ribosome assembly. Previous data have shown that depletion of each ribosomal protein has a specific effect on the pre-rRNA processing pathway for the large subunit. However, depletion of an essential ribosomal protein may cause destabilization of this multi-component system and abort assembly. Thus, truncation of eukaryote-specific extensions has the potential to reveal more specific functions of r-proteins. We have systematically truncated and introduced single mutations in the eukaryote-specific extensions of six r-proteins (L4, L6, L7, L8, L25, L35) in Saccharomyces cerevisiae 60S subunits. While in vivo expression of some truncated r-proteins resulted in depletion phenotypes, we were able to identify several mutants with distinct defects. Our approach promises to assign specific roles to the eukaryote-specific extensions of r-proteins in ribosome assembly and reveal the significance of intrinsically disordered domains in processing and folding of rRNAs, as well as binding of r-proteins.
Keywords: ribosome assembly , ribosomal proteins
Abstract:
RNase P is an essential enzyme that catalyzes the 5’-maturation of precursor tRNAs (pre-tRNAs) in a Mg2+-dependent reaction. While a proteinaceous RNase P is found in several eukaryotes, ribonucleoprotein (RNP) variants have been found in all three domains of life. The RNP forms consist of a catalytic RNA (RPR) and a variable number of protein subunits (RPPs): one in Bacteria, ≤ 5 in Archaea, and ≤ 10 in Eukarya. While the RPR alone is catalytically active at high Mg2+ concentrations, addition of the protein cofactor enables the RPR to function at near-physiological conditions by aiding substrate recognition and active site metal ion affinity, as best exemplified by studies on bacterial RNase P. We reported thematic parallels in archaeal RNase P, composed of an RPR + two RPP heterodimers and a lone RPP (POP5-RPP30, RPP21-RPP29 and L7Ae). Addition of POP5-RPP30 increases the RPR’s rate of pre-tRNA cleavage by 60-fold, while RPP21-RPP29 enhances the RPR’s substrate affinity by 15-fold (1). However, little is known about how these archaeal RPPs assemble with the RPR to accomplish their functional roles. We have employed a site-directed hydroxyl-radical footprinting strategy to map the binding sites of each RPP on the Pyrococcus furiosus (Pfu) RPR and a pre-tRNA, building on earlier studies with Escherichia coli RNase P (2) and recent mapping of Pfu L7Ae on the Pfu RPR (unpublished). Here, we constructed single Cys-substituted mutant derivatives of POP5 and RPP29 in which residues at or near their proposed substrate- and RPR-binding interfaces, respectively, were modified to Cys. These mutant derivatives were then purified and modified with an iron complex of EDTA-2-aminoethyl 2-pyridyl disulfide (EPD-Fe) to form site-specific protein-tethered nucleases. Reconstituted holoenzymes (RPR + 5 RPPs, including modified POP5 or RPP29) in the absence or presence of pre-tRNA were incubated with hydrogen peroxide and ascorbate to promote OH·-mediated cleavages at proximal sites (< 10 Å) on either the Pfu RPR or the pre-tRNA substrate, which were then mapped. The intra-molecular RPP-RPR and RPP-substrate distance constraints from these ongoing studies are expected to lead to a molecular model of the holoenzyme and provide insights into protein-aided RNA catalysis in archaeal RNase P.
References:
(1) Chen WY, Pulukkunat D, Cho IM, Tsai HY, Gopalan V. (2010) Dissecting functional cooperation among protein subunits in archaeal RNase P, a catalytic ribonucleoprotein complex. Nucleic Acids Res. 38: 8316-8327.
(2) Biswas R, Ledman D, Fox RO, Altman S, Gopalan V. (2000) Mapping RNA-protein interactions in ribonuclease P from Escherichia coli using disulfide-linked EDTA-Fe. J Mol Biol. 296: 19-31.
Keywords: archaeal RNase P, POP5 and RPP29, hydroxyl-radical footprinting
Abstract not available online - please check the printed booklet.
Abstract:
The long-non-coding Steroid Receptor Activator RNA (SRA lncRNA) is part of a SRC-1 enzyme complex that acetylates histones.1 This complex permits the transition of chromatin regions from silenced heterochromatin to expressed euchromatin.2,3 The SRC-1/SRA complex enhances the expression of genes controlled by steroid nuclear receptors.4,5,6 SRA1 RNA also interacts with many proteins, DDX17,6 SHARP,7 and others.8 Nuclear receptors are known to stimulate certain cancers; interestingly SRA RNA is dramatically overexpressed in, and stimulatory to, tumor cells.9 The SRA lncRNA facilitates chromatin remodeling as a non-coding RNA rather than as an mRNA. However, SRA is alternatively spliced into an mRNA encoding the SRA1p protein.10 Thus the gene for SRA is considered both an mRNA and lncRNA coding gene. SRA1p, along with SLIRP and SHARP, are repressors of SRA action and thus important checks on differentiation11 and tumorigenesis. There are no structures of these proteins bound to RNA and the evidence for direct interactions is based on immunoprecipitation. Our NMR structure of the c-terminal domain of hSRA1p, previously thought to be an RRM domain, demonstrates that it is not an RRM nor does it have apparent SRA RNA binding activity. In contrast, RRM domains from SHARP and SLIRP are clearly identifiable and our progress towards the NMR structures of these RNP complexes will be presented.
References:
1. Lanz RB, et al. Tsai SY, Tsai MJ, O Malley BW (1999) Cell 97: 17-27.
2. Onate SA, et al. (1995) Science 270: 1354–7.
3. Onate SA, et al. Edwards DP, O Malley BW (1998) JBC 273: 12101–8.
4. Alen P, et al. (1999) MCB 19: 6085–97.
5. Aarnisalo P, et al. (1998) PNAS 95: 2122–7.
6. Watanabe M, et al. (2001) EMBO J. 20: 1341–52.
7. Shi Y, et al. (2001) Genes Dev. 15: 1140–51.
8. Colley SM, Leedman PJ (2011) Biochimie 93:1966-72.
9. Lanz RB, et al. (2003) MCB 23: 7163-76.
10. Chooniedass-Kothari S, et al. (2004) FEBS Lett. 566: 43–7.
11. Hubé F, et al. (2011) NAR 39: 513-25.
Keywords: NMR, lncRNA, epigenetics
Abstract:
Alternative splicing of the HIV-1 genome is necessary for translation of the complete viral proteome. Host proteins, such as hnRNP A1, are used to regulate splicing at the various donor and acceptor sites along the viral genome. One such site regulated by hnRNP A1 is the conserved 3’ acceptor splice site A7 (ssA7). Silencing of splicing at this site is necessary in order to retain the Rev Responsive Element (RRE) in the adjacent tat/rev intron. The RRE is responsible for nuclear export of unspliced and partially spliced transcripts. .
Our research seeks to clarify the binding determinant of hnRNP A1 on ssA7 by developing a structural model that will correlate ssA7 structure to its splicing function. For this model we are using isolated domains of both hnRNP A1 and ssA7. The protein UP1 is composed of the two RRM domains of hnRNP A1. For ssA7, SL3 of the three stem loop structure is examined as this contains a high affinity UAG binding site for hnRNP A1. We previously solved the 3D solution structure of SL3 by NMR and found the UAG is located in a terminal heptaloop.
Two separate pathways are being pursued for the development of the UP1:ESS3 co-structure: X-ray crystallography and HADDOCK modeling. For the X-ray crystallography, conditions for obtaining crystals of the co-structure are currently being optimized.
HADDOCK is “an information-driven flexible docking approach for the modeling of biomolecular complexes.” We are deriving the points of contact between UP1 and ESS3 with both NMR and mutagenesis experiments. With NMR, NOEs give exact points of contact between the two molecules while chemical shift changes show which residues might be involved actively or passively in the binding. Residues involved in binding can also be identified through mutagenesis experiments that alter residues on either UP1 or ESS3. Isothermal Titration Calorimetry (ITC) experiments are performed to determine the effect of the mutation on the binding of the two molecules.
References:
Levengood, J.D., Rollins, C., Mishler, C. H. J., Johnson, C.A., Miner, G., Rajan, P., Znosko, B.M., and Tolbert, B.S. (2012) Solution Structure of the HIV-1 Exon Splicing Silencer 3. J. Mol. Biol. 415:680-698.
Keywords: RRM, Splicing, Structure
Abstract:
RNA processing enzymes and catalytic RNAs perform essential roles throughout biology and are often the targets of therapeutic intervention for treating human disease. Structure-function studies of RNA binding and RNA-processing reactions, in which the effects of specific variations in sequence on specific reaction parameters such as binding kinetics, equilibrium binding affinity and catalytic rate, have provided deep insights into biological function to be gained. Nonetheless, our perspective is severely limited by the relatively small number of sequence variants that can be analyzed. Using RNase P processing of pre-tRNA as an experimental system are developing a set of tools based on high-throughput sequencing and competitive substrate kinetic analysis to accurately and simultaneously determine kinetic and equilibrium binding constants for large RNA substrate. Next generation sequencing is used to follow the distribution of sequences in substrate and product populations as a function of reaction progress. Competitive substrate kinetic analyses are used to calculate rate constants for all members of the substrate population from these data. For RNase P the resulting high-density structure-function data sets are providing unique insights into patterns of molecular recognition and the nature of specificity in RNA-protein interactions. Although powerful, an inherent limitation of competitive multiple turnover kinetics is that product inhibition, inactive substrate populations and multiphasic kinetics can limit precision. Using single turnover reactions which conform more directly to simple exponential kinetics should allow high resolution data sets to be gained for both binding kinetics and effects on catalysis. A similar approach is being developed to determine equilibrium binding constants by analyzing the distribution of sequences in free and bound populations analyzed using simple competitive binding models. Here, free and bound populations are separated and purified by native gel electrophoresis. In combination these approaches are providing a comprehensive understanding of how substrate sequence and structure affect binding affinity, association kinetics and catalysis.
Keywords: RNA enzymology, high throughput sequencing, equilibrium binding
Abstract:
In post-transcriptional regulation, many proteins and/or microRNAs bind to an RNA molecule to modulate its biological functions. Such process comprises multiple binding reactions, and in order to enable combinatorial gene regulation it is necessary that these binding partners of RNA communicate with each other, or in other words that their binding to the RNA exhibits cooperativity. Even in the absence of direct physical interactions between the binding partners, such cooperativity can be mediated through the secondary structure of the RNA molecule, since the secondary structure affects accessibility to the various binding sites. Here we propose a quantitative measure of this structure-mediated cooperativity that can be calculated for an arbitrary RNA sequence using computational secondary structure prediction methods. Focusing on an RNA with two binding sites, we derive a characteristic free energy difference as the measurement for describing how the binding strength of one site is affected by the occupancy of the other one. We apply this measurement to a large number of human mRNA sequences, and find that structure-mediated cooperativity is a generic feature of mRNA sequences. Most interestingly, this structure-mediated cooperativity not only affects binding sites in close proximity along the sequence but also configurations in which one binding site is located in the 5'UTR and the other binding site is located in the 3'UTR of the molecule.
Keywords: RNA-protein interactions, cooperativity, RNA structure
Abstract:
In recent years, long noncoding RNAs (lncRNAs) have been increasingly recognized as important regulators of gene expression in various levels. The human genome encodes thousands of lncRNAs, and an increasing number of these have been associated with human diseases. lncRNA structures and lncRNA-protein interactions are expected to play essential roles in their gene gene regulatory functions, however, our knowledge about that is limited. To address this issue, we have developed a novel method for high-throughput probing of RNA structure using massively parallel sequencing (Mod-seq). Compared to traditional RNA structure probing methods, Mod-seq provides substantial increases in throughput, allowing rapid and simultaneous probing of the whole transcriptome. In addition, Mod-seq results in quantitative analysis of lncRNA structure. By comparing Mod-seq results in vivo and in vitro, we aim to identify putative lncRNA-protein interactions.
Keywords: RNA Structure, lncRNA
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
Analysis of the role of a long non-coding RNA in cancer
Mengdi Liu and Saba Valadkhan
Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, OH 44106
Long non-protein-coding RNAs (lncRNAs) are pervasively transcribed throughout eukaryotic genomes. Supporting the biological relevance of these transcripts, multiple studies have shown that significant numbers of long ncRNAs are regulated during development and exhibit cell type-specific expression, localize to specific subcellular compartments and are associated with human diseases. It has been shown that the expression of a number of lncRNAs is perturbed in cancer, suggesting that some lncRNAs may be important in the process of malignant transformation or other aspects of biogenesis of cancers.
To gain further insight into the role of lncRNAs in human cancers, we analyzed the expression level of a long intergenic RNA (BORG) in human lung cancer tissues, adjacent normal tissues and different cancer cell lines. We are in the process of defining the impact of plasmid-mediated overexpression and shRNA-mediated knockdown of this lncRNA in NMuMg, NME and D2 breast cancer cell lines. These studies will elucidate the possible function of a lncRNA in malignant transformation and cancer progression.
Keywords: lnc RNA, Cancer
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
RNA structure and ribonucleoprotein (RNP) complex formation are critical for eukaryotic gene expression. One class of enzymes called DEAD-box RNA helicases play fundamental roles in remodeling RNA and RNP structure in every aspect of RNA metabolism. Nevertheless, how the biochemical activity of these enzymes is connected to the biological function remains elusive. To define the precise roles of individual DEAD-box proteins, our laboratory recently demonstrated that DEAD-box protein Dbp2 functions in nuclear gene expression steps in Saccharomyces cerevisiae. We will present evidence that Dbp2 interacts physically with the mRNA binding protein Yra1. Moreover, we will show that Dbp2 is required for efficient assembly of mRNA-binding proteinYra1, Nab2, and Mex67 onto poly(A)+ RNA. In addition, we will demonstrate that Dbp2 is an active RNA helicase in vitro and that Yra1 reduces the unwinding activity of Dbp2. We propose that Dbp2 unwinds secondary structure within mRNA to facilitate efficient messenger ribonucleoprotein (mRNP) assembly. Once the mRNP is properly assembled, Yra1 then inhibits Dbp2 to prevent further rearrangement of the mRNP. This provides a novel mechanism for regulation of DEAD-box proteins in RNP assembly.
Keywords: DEAD-box protein, mRNP assembly, mRNA binding protein
Abstract:
RNase P is an essential ribonucleoprotein (RNP) enzyme that cleaves the 5’-leader of precursor tRNAs in a Mg2+-dependent manner to produce functional mature tRNAs in all three domains of life. RNase P present in the thermophilic archaeon Pyrococcus furiosus contains one catalytic RNA (abbreviated RPR for RNase P RNA) and five protein cofactors (RPPs) called POP5, RPP21, RPP29, RPP30, and L7Ae. RNase P activity (of varying potency) can be demonstrated in the holoenzyme reconstituted in vitro using the RPR with either or both interacting RPP pairs (POP5−RPP30 or/and RPP21−RPP29) and with all five RPPs. Previous studies have shown that (1) these RPP pairs can be co-expressed and co-purified from Escherichia coli, (2) POP5−RPP30 forms a tetramer, and (3)RPP21−RPP29 forms a dimer (in the absence of the cognate RPR). However, the stoichiometry of the individual subunits in the reconstituted RNase P complex remains unknown. Native mass spectrometry, ion mobility and tandem mass spectrometry were employed to explore the interaction of the two RPP pairs by themselves, with each other and with the RPR. Our results confirmed that the two RPP pairs behave as reported previously, and additionally revealed a higher-order structure (POP52−RPP302.RPP21−RPP29). However, in the presence of the RPR, only one copy of each RPP is found in the holoenzyme complex. Moreover, we observed that surface induced dissociation (SID) can break up the holoenzyme (lacking L7Ae), while collision induced dissociation (CID) cannot do so effectively. Our studies collectively demonstrate the advantage of SID in successfully determining the composition of an RNP complex and provide insight into the assembly of archaeal RNase P.
Keywords: RNase P, mass spectrometry, stoichiometry
Abstract not available online - please check the printed booklet.
Abstract:
Thg1 and Thg1-like proteins (TLPs) are found in Eukarya, Archaea, and Bacteria. Thg1 is a part of a novel class of enzymes that catalyzes 3&prime to 5&prime nucleotide addition to tRNAHis as the final essential maturation step of this tRNA. Other Thg1 family members utilize 3&prime - 5&prime polymerase activity to repair the 5&prime -ends of tRNA during 5&prime -tRNA editing, however, additional unidentified roles for these enzymes in biology are likely.
The crystal structure of human Thg1 (hThg1) complexed with two molecules of GTP confirmed that hThg1 utilizes a two-metal ion mechanism for G-1 addition and provided evidence for the binding site for the activating NTP; however, it did not provide any information on Thg1 interaction with tRNAHis . Previous work identified critical residues for G-1 addition, including several residues (D68, H152 and N198) that appear to play a role in tRNA binding and/or recognition, according to biochemical assays. However, the molecular basis for the involvement of these residues and others in binding to tRNAHis is not known.
Nuclease footprinting is being used to provide approximate regions of tRNAHis that may be involved in hThg1 binding. At present, nuclease digestion conditions have been optimized. Preliminary nuclease footprinting experiments suggest that there are some protected regions of tRNAHis in the presence of hThg1. However, the use of a nuclease-based method to determine RNA-protein contacts has limitations including abrogation of hThg1 binding to tRNAHis. Furthermore, bulky nucleases are more sensitive to steric hindrance, especially in the presence of protein. Additionally, ribonuclease cleavage could alter the structure of the RNA and cause secondary cleavages. Therefore, additional complementary approaches will also be employed to investigate and strengthen conclusions from these data.
Keywords:
Abstract:
2’-O-ribose of all ribosomal RNA (rRNA) is methylated and it is the most common covalent modification. Previously we showed a critical role of L13a in rRNA methylation and inhibition of rRNA methylation does not affect global protein synthesis in mammalian cells [1]. However, our subsequent studies identified a novel role of rRNA methylation for the translation of a cohort of Cellular Internal Ribosome Entry Sites (IRESs) [2]. Recently, using L13a protein as a model we have initiated a study investigating the mechanism of ribosomal protein incorporation in mammalian ribosome. This study has identified a critical residue of L13a essential for rRNA binding [3]. The extra-ribosomal activity of L13a in IFN-gamma induced translational silencing relies on its release from 60S ribosomal subunit. However, the mechanism of its release and how the released L13a assembled into the silencing competent RNA-binding complex and recognized by the target mRNAs harboring GAIT element in the 3’UTRs is poorly understood. To understand the specific domains or amino acid residues responsible for this diverse activity of a single ribosomal protein, we have conducted extensive deletion and mutational analysis of this protein. The result of this analysis shows significant insights about the diverse activity of this protein.
References:
[1]. Chaudhuri et al. RNA. 2007, 13(12): 2224-2237.
[2]. Basu et al. Mol. Cell. Biol. 2011, 31(2): 4482-4499.
[3]. Das et al. Mol. Cell. Biol. 2013, 33(15): 2829-2842.
Keywords: Ribosomal Protein L13a, Ribosome Biogenesis, Translational Silencing
Abstract:
Mitochondrial mRNAs in kinetoplastid parasites require extensive remodeling by a unique process termed uridine insertion/deletion RNA editing. For the majority of transcripts, RNA editing is required to create translatable open reading frames, and thus editing is essential for proliferation of both human and insect vector stages of the kinetoplastid, Trypanosoma brucei. Small trans-acting guide RNAs (gRNAs), encoded in the minicircle component of the mitochondrial genome, direct proper U insertion or deletion. Editing is catalyzed by the multiprotein RNA editing core complex (RECC) or 20S editosome, although additional components are emerging as equally critical to the editing process. We have recently characterized a dynamic multiprotein complex termed MRB1 (mitochondrial RNA binding complex 1) as a key component of the editing machinery. We reported the presence of an MRB1 core that contains at least 6 proteins and is essential for RNA editing initiation. We have also identified subcomplexes containing the TbRGG2 RNA binding/annealing protein; TbRGG2 subcomplexes are essential for 3’ to 5’ progression of editing on pan-edited RNAs. Numerous additional proteins interact in a substoichiometric manner with the MRB1 core and TbRGG2 subcomplexes, and their functions are as yet unknown. Here, we present ongoing studies of one such protein, MRB1590. RNAi-mediated knockdown of MRB1590 in insect stage T. brucei leads to a modest growth defect and a specific inhibition of the 3’ to 5’ progression of editing of A6 mRNA that may reflect a role in efficient utilization of one or a few gRNAs. Structural analysis shows that MRB1590 is an ABC ATPase domain with similarity to proteins involved in DNA repair. In vivo pulldown studies demonstrate RNA-dependent interactions between MRB1590 and both TbRGG2 and the GAP1/2 component of the MRB1 core. Interestingly, MRB1590 interacts in an RNA-independent manner with the Zn finger protein, MRB6070, suggesting the presence of another multiprotein component of the dynamic MRB1 RNA editing complex, which may contain editing specificity factors. These studies provide another piece in the puzzle of our ongoing characterization of the essential and multifunctional RNA editing complex, MRB1.
Keywords: RNA editing
Abstract not available online - please check the printed booklet.
Abstract:
A library screening approach identified naturally occurring sequences for an RNA G-quadruplex binding ligand
Abstract
G-quadruplex (GQ) structures are secondary structures formed by both RNA and DNA. It consists of four guanines joined by Hoogsteen base pairings and stabilized by a monovalent cation.1 RNA G-quadruplexes have gained an increasing attention due to their critical roles in translational regulation of many important genes such as VEGEF, NRAS, FMRI and etc.2,3,4 Although designing of small molecules to target RNA GQs have become an important therapeutic approach, it is difficult to achieve the selectivity among GQs formed by the various RNA sequences. Therefore, it is crucial to identify all possible GQs in the human transcriptome that can potentially bind to these small molecules. A pilot study was performed to address this issue. We designed an RNA sequence library which has the possibility to form thousands of different GQ structures. Then we screened this library against kanamycin which is known to bind with RNA secondary structures including GQs.5 We found seven different RNA sequences that bind selectively with kanamycin and three of them are found in human transcriptome. The formation of GQ by each RNA sequence was confirmed by circular dichroism (CD), RNase T1 footprinting and CD melting. The binding affinities of kanamycin with the selected RNAs were compared using fluorescence Intercalator displacement assay (FID) where a fluorescence dye is replaced from the GQ by titrating with kanamycin.6 These studies show the possible GQ targets for kanamycin and how tightly such sequences interact with it. Therefore our studies showed the importance of this library screening approach to identify possible G-quadruplex targets for a small molecule which can be used as therapeutic. Elaborating the pool of the G-quadruplex library will be a further improvement of this approach to identify all potential targets in human transcriptome for a given small drug molecule.
References:
References
1. S. Neidle and Balasubramanian, S. , RSC Biomolecular Sciences., 2006.
2. J. C. Darnell, K. B. Jensen, P. Jin, V. Brown, S. T. Warren and R. B. Darnell, Cell, 2001, 107, 489-499.
3. M. J. Morris, Y. Negishi, C. Pazsint, J. D. Schonhoft and S. Basu, J. Am. Chem. Soc., 2010, 132, 17831-17839.
4. S. Kumari, A. Bugaut, J. L. Huppert and S. Balasubramanian, Nat. Chem. Biol., 2007, 3, 218-221.
5. O. Aminova, D. J. Paul, J. L. Childs-Disney and M. D. Disney, Biochemistry, 2008, 47, 12670-12679.
6. D. Monchaud, C. Allain and M. P. Teulade-Fichou, Bioorg. Med. Chem. Lett., 2006, 16, 4842-4845.
Keywords: RNA G-quadruplex, G-quadruplex Ligands , Library Screening
Abstract:
Juvenile myoclonic epilepsy (JME) is a common form of idiopathic generalized epilepsy, with a strong genetic pathophysiology. Genes associated with JME have only recently been discovered; yet the contribution of these genes to JME have not been elucidated outside of family-based linkage analysis. We focus on understanding how one gene identified through linkage analysis, BRD2, is regulated and altered in JME; specifically, how changes in the alternative splicing of BRD2 affect its expression. The importance of proper BRD2 expression is highlighted in mouse models, where Brd2 null mice lack proper CNS development and heterozygous Brd2+/- mice show hypersensitivity to induced seizures. We predict that a potential source of BRD2 mis-regulation in JME patients involves differential inclusion of the ultra-conserved poison exon 2a. Exon 2a inclusion in BRD2 pre-mRNA is thought to cause premature translation termination. Using cycloheximide and caffeine, both of which are known to inhibit nonsense mediated decay (NMD), we show that transcripts containing exon 2a are targets for NMD in tissue culture. To understand the effect of mutations on exon 2a inclusion, we focused on characterizing the splicing impact of polymorphic regions in BRD2 that show high association with JME in linkage studies. Using a BRD2 minigene system in tissue culture, we show increased exon 2a inclusion when shortening a GU repeat region in intron 2. Furthermore, overexpression or inhibition of a splicing regulator, CELF1 which binds GU-rich elements and is associated with epilepsy in mouse models, modulates endogenous BRD2 exon 2a inclusion. These initial studies suggest a role for BRD2 alternative splicing in the etiology of JME, and future studies will focus on how mutations found in patient samples affect exon 2a inclusion. We hope to use these findings to engineer targeted therapeutics using antisense oligonucleotide technology in order to one day modulate BRD2 splicing and expression in JME.
Keywords: BRD2, Ultraconserved Exons, NMD Gene regulation
Abstract:
Steriod Receptor RNA Activator (SRA) RNA is an RNA with dual roles, acting as both a ncRNA and as a template for a protein (SRAP)1. The protein that the SRA RNA codes for produces a two domain protein with one of the proposed domains acting as an RNA recognition motif due to the presence of the RNA recognition sequence LLVQEL. The second domain is also implicated to serve as a nuclear localization signal2. Previous studies have implicated that the STR7 region of the SRA RNA and SRAP are associated with each other and may be part of the same complex3. SRA RNA and SRAP have been implicated in a wide variety of functions and interactions in vivo. There is particular interest in the role of SRAP in ER+ breast tissue tumors. Studies have shown increased amounts of SRAP within these tumors, however the clinical prognosis of such patients is mixed4,5. Further studies to elucidate the exact mechanism of SRAP’s function are required. Presented here is the structure of the human SRAP of the 2nd domain of the protein via nuclear magnetic resonance (NMR). In addition to the 2nd domain's structure, studies of the full length protein and domain 1 via NMR will be presented. Due to the previous research indicating that the SRA RNA and SRAP are associated with each other, chemical shift perturbation (CPS) with the STR7 region of the RNA will also be discussed.
References:
Chooniedass-Kothari, S., Emberly E., Hamedani, M.K., Troup, S., Wang, X., Czosnek, A.; Hube, F.; Mutawe, M.; Watson, P.H., Leygue, E. FEBS Lett 2004, 566, 43-47
Leygue, E. Nucl Recept Signal 2007 5, e006
Hube, F.; Velasco, G., Rollin J., Furling, D., Francastel C. Nuc Acid Res 2011, 39(2) 513-525
Yan, T., Penner, C.C., Skliris, G.P. , Cooper, C., Nugent, Z., Blanchard, A., Hamedani, M.K., Wang X., Myal, Y., Murphy L.C., Leygue, E. J Cancer Res Clin Oncol 2013, 139(10) 1637-1647
Chooniedass-Kothari, S.; Hamedani, M. K.; Troup S.; Hubé, F.; Leygue, E. Int J Cancer 2006, 118, 1054-1059.
Keywords: SRAP, NMR, cancer
Abstract not available online - please check the printed booklet.
Abstract:
The HIV-1 Dimerization Initiation Sequence (DIS) is a conserved hairpin motif in the 5’-UTR of its RNA genome. DIS plays an important role in genome dimerization by formation of a “kissing complex” (KC) between two homologous hairpins. Dimerization of HIV-1 genomic RNA is facilitated by Nucleocapsid protein, a small, basic protein with two zinc-finger domains Under-standing the kinetics of this interaction is key to exploiting DIS as a possible HIV drug target. Here, we present a single-molecule Förster Resonance Energy Transfer (smFRET) study of dimerization kinetics. Our data show the real-time formation and dissociation dynamics of individual kissing complexes, as well as the rearrangement of the kissing complexes into extended duplexes. Interestingly, the single-molecule trajectories reveal the presence of a previously unobserved bent intermediate required for extended duplex formation. Mutation of universally con-served residue A272 leads to dramatically altered behavior, showing that this adenosine is essential for the formation of this intermediate,. The intermediate state is stabilized by Mg2+, but not by K+ cations. We propose a 3D model of a possible bent intermediate and a minimal dimerization pathway consisting of three steps including two obligatory intermediates (kissing complex and bent intermediate) and driven by Mg2+ ions. We have also shown that certain aminoglycosides with high ionic charge (Neomycin, Paromomycin and Lividomycin) mimic the function of Mg2+. This displacement results in a stabilized KC and stalling the dimerization pathway at KC stage. HIV-1 nucleocapsid protein (NCp7) facilitates formation of the extended RNA duplex by shifting the KC and monomer RNA equilibrium toward the KC. Furthermore, experiments with NCp7 support the conclusion that the proposed newly observed bent dimer conformation is an on-path obligatory intermediate.
Keywords: smFRET, Aminoglycosides, Nucleocapsid Protein
Abstract not available online - please check the printed booklet.
Abstract:
Escherichia coli RfaH, a paralogue of general transcription factor NusG, activates expression of long, poorly translated, horizontally transferred virulence operons which are potential targets of Rho-mediated polarity. RfaH consists of two domains connected by a flexible linker. The N-terminal domain is recruited to the RNA polymerase halted at a specific ops site and reduces transcription pausing and termination thereafter. The C-terminal domain interacts with the ribosomal protein S10. By physically linking the RNA polymerase to the translational machinery, RfaH may (i) enable ribosome recruitment during initiation and (ii) couple transcription and translation during elongation. The first activity may be particularly important because RfaH-controlled operons do not possess optimal Shine-Dalgarno (SD) elements. Indeed, deletion of the SD element from a model operon confers strong dependence on RfaH. Although many bacterial operons share the same feature, the mechanism of non-SD led translation initiation remains unclear, and deciphering the mechanism of RfaH could provide potential insights into this hitherto unknown cellular process.
Here, we investigate the contribution of leader region features to RfaH mediated translation activation using a reporter in which the ~200 nt rfb operon leader region with the ops site located in the middle is followed by the lux operon. First, we show that RfaH has roughly the same effect on lux expression when the spacing between the ops and the translation start is varied from 44 to 435 nts, suggesting that sequences around the ops and the translation initiation region are the most important. Second, we found that RfaH does not influence the start site selection by the ribosome. This may imply a start codon scanning mechanism by the 30S subunit captured by RfaH bound to the leading RNAP. We also show that the ribosomal protein S1, which acts as a translation enhancer in the absence of SD, is not required for RfaH-mediated activation. Thus RfaH appears to be the only component required for the potential coupling and translation of its target operons. In the future, we will dissect the fine details of the leader region and carry out footprinting experiments to map the site of the ribosome recruitment by RfaH.
Keywords: RfaH, Translation, Ribosome
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
High Throughput Sequencing (HTS) techniques are useful in the study of RNA Biology, providing relatively fast and inexpensive genome-wide coverage. However, they produce unique challenges, most notably in the large volumes of information generated, and in the fragmentation of RNA reads across exon boundaries. There exist well established tools for global differential expression analysis using RNA-Seq, but there are many other applications of HTS in RNA Biology, where the local coverage along the transcripts is of interest, such as, e.g., ribosome profiling. Here we introduce computer programs and techniques developed to analyze such specialized HTS data sets. In particular, we share techniques used to, from an aligned data set, remove contaminant reads (such as rRNA in a ribosome profiling study), visualize genome-wide coverage, aggregate reads by transcripts, normalize data to different standards, calculate coverage ratios, handle biological replicates, and quantify and identify changes in read distribution along transcripts, both globally and transcript by transcript. One benefit of these tools is that they can be equally applied to whole transcriptome data sets (such as TrueSeq), to ribosome profiling data sets, or to other data sets resulting from special purpose library construction schemes involving RNA. We also discuss some of the quality control mechanisms developed to test wet lab effectiveness and program validity.
Keywords: Computation, High Throughput Sequencing, Ribosome Profiling
Abstract:
Liver, the major metabolic organ of the body, undergoes dramatic transitions with regards to structure and function during development. Alternative mRNA splicing (AS) is one of the most prominent mechanisms to generate mRNA complexity, which in turn results in increased proteome diversity. Therefore, we aim to investigate the functional role of AS in mammalian liver development.
To characterize the conserved AS program during liver development, we performed RNA-seq of mouse livers between E18 and P28 timepoints. Among the 150 validated events, 108 (72%) exhibit an increase, and 42 (28%) exhibit a decrease in inclusion of the variably spliced region. Based on this dataset, we performed a temporal analysis of intervening time points, (E16, E18, P0, P2, P7, P14, P28 and P90) and have characterized events that follow prenatal, postnatal and biphasic patterns of splicing. Direct comparison of 118 splicing transitions between mouse and human shows that 57 are evolutionarily conserved during development. Studying these splicing transition networks will be fundamental to understand the regulatory programs that govern development.
We investigated the expression of MBNL1 and CELF1 during liver development and observed that they are strongly down regulated (13 fold and 6 fold respectively) during the first four weeks of liver development. This indicates that changes in splicing factors might play a major role in governing the AS landscape during development. Using knockout and transgenic mice we will further investigate RBPs role in liver development by analyzing the change in splicing patterns due to the absence or presence of these factors. Further studies plan to perform genome-wide iCLIP experiments to determine the direct pre-mRNA targets of these splicing factors.
This study will help us identify conserved mRNA processing transitions in liver development and also establish strong correlations between splicing changes and it’s putative regulators.
Keywords: Alternative Splicing, Liver Development, RNA Binding Proteins
Abstract not available online - please check the printed booklet.
Abstract:
Nuclear speckles are dynamic subnuclear accumulations of pre-mRNA splicing factors and other co-transcriptional processing factors often found nearby active transcription sites (1). The nuclear speckle splicing factor called Son has within its sequence a cluster of unique tandem repeats as well as an arginine-serine-rich (RS) domain, a G-patch and a double-stranded RNA binding domain at its C-terminus. Son’s unique repeats are required for proper nuclear speckle organization (2), and other domains are important for splicing regulation for a subset of human mRNAs (3,4). To investigate Son’s activities at transcription sites in situ, we used the U2OS 2-6-3 cell line containing an integrated transgene array that allows visualization of gene expression at a specific site of chromosome 1 (5). Our lab discovered that unlike other SR proteins that are recruited to this reporter array after transcriptional activation, Son localizes to the inactive reporter gene locus and is absent following transcription induction. Immunofluorescence analysis shows that upon transcriptional activation, enrichment of Son at the U2OS 2-6-3 locus decreases within thirty minutes and leaves the locus within sixty minutes. SR protein alternative splicing factor 2 (ASF/SF2) is not present at the inactive locus and is recruited to the active locus after Son clears the locus. Chromatin immunoprecipitation with anti-Son antibodies (Son-ChIP) indicates that Son enrichment is highest at the promoter region of the U2OS 2-6-3 transgene array. We hypothesize that Son maintains the heterochromatic state of the inactive reporter array, and that Son may be needed during the first steps of gene activation to recruit components required for mRNA synthesis and/or processing. Heterochromatin maintenance would be a novel role in gene expression for a splicing factor. Experiments to determine Son’s specific role in gene expression at this locus as well as endogenous human gene loci are being pursued.
References:
1) Spector, D. L. and Lamond, A. I. (2010). Cold Spring Harb Perspect Biol. doi: 10.1101/ cshperspect.a000646
2) Sharma, A., Takata, H., Shibahara, K., Bubulya, A., and Bubulya, P. A. (2010). Mol. Biol. Cell. 21, 650-663.
3) Sharma, A., Markey, M., Torres-Muñoz, K., Varia, S., Kadakia, M., Bubulya, A., and Bubulya, P. A. (2011). J. Cell Sci. 124, 4268-4298.
4) Ahn, E. Y., Yan, M., Malakhova, O. A., Lo, M. C., Boyapati, A., Ommen, H. B., Hines, R., Hokland, P., and Zhang, D. E. (2011). Molecular Cell. 42, 185–198.
5) Janicki, S. M., Tsukamoto, T., Salghetti, S. E., Tansey, W. P., Sachidanandam, R., Prasanth, K. V., Ried, T., Shav-Tal, R., Bertrand, E., Singer, R. H., and Spector, D. L. (2004). Cell. 116, 683-698.
Keywords: heterochromatin, gene expression, Son
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
A universal core secondary structure for ribosomal RNA (rRNA) has been identified across kingdoms of life. (1,2) However, yeast rRNA, and eukaryotic rRNA in general, have extra blocks of sequences relative to that of prokaryotic rRNA, called “expansion segments” (ES). These ES vary in their length and sequence, both within and among different organisms. This variability seems to preclude ES from playing crucial roles in the function of the ribosome. (3)
Isolated attempts to understand the function of ES have been made before. Previous reports indicate that some, but not all ES can tolerate insertions. (4) Also, deletion mutational analysis of two ES has been shown to affect ribosome biogenesis (5). Despite these reports, a thorough study of the precise roles of these ES remains to be done, possibly because of the limitations in the availability of convenient systems to study rRNA mutants.
We set out to systematically investigate the potential roles played by expansion segments of the 25S rRNA in Saccharomyces cerevisiae ribosome biogenesis. We began with deleting five of the eukaryote-specific ES in yeast large subunit rRNA. We used a temperature sensitive PolI mutant yeast strain in which transcription of rDNA is driven from a plasmid-borne rDNA copy with a GAL promoter (6). Also, we developed yeast strains derived from this strain in which one can pull down pre-ribosomes using affinity purification. The phenotype of the mutants was first assayed by studying their growth. Following this, primer extension assays and affinity purifications were used to zoom in on the precise ribosome assembly phenotype that these mutants exhibit.
This systematic study will help us unravel the yet unexplored functions of these eukaryote-specific ES and pave the way for a deeper understanding of the mechanisms of ribosome biogenesis in general.
References:
1. Veldman, G.M., et al. (1981) Nucleic Acids Res. 9:6935-6952
2. Clark, C. G., et al. (1984) Nucleic Acids Res. 12: 6197-6220.
3. Gerbi, S.A. (1996). In ‘Ribosomal RNA Structure, Evolution, Processing, and Function in Protein Biosynthesis’, R.A. Zimmerman, and A.E. Dahlberg, eds. (Boca Raton, FL, CRC Press), pp. 71-87
4. Musters, W., et al. 1989) Mol. Cell. Biol. 9:3989-3996
5. Jeeninga, R. E., et al. (1997). RNA. 3: 476-488
6. Nogi, Y., et al. (1991). Proc. Natl. Acad. Sci. USA 88: 3962-3966
Keywords: rRNA , ribosome biogenesis, expansion segments
Abstract not available online - please check the printed booklet.
Abstract:
Alternative splicing of the human immunodeficiency virus (HIV) genome is a carefully orchestrated event with a strong impact on both function and infectivity. This splicing is accomplished by the cis RNA regulatory elements interacting with trans host regulatory proteins. One such cis regulatory element is the third stem loop of acceptor splice site A7, termed exon splicing silencer 3 or SLESS3. Utilization of this splice site in combination with donor splice site D4 produces tat and rev mRNA. Although SLESS3 has been shown to interact with the UP1 domain of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) to block the assembly of the spliceosome, the mechanism of binding has yet to be determined. Biophysical studies have been implemented to further the understanding of this interaction, through mutational analysis, isothermal titration calorimetry, and two dimensional NMR. These studies provide insight into the elements of both SLESS3 and UP1 that contribute to the formation of this RNA-protein complex.
Keywords: Alternative Splicing, NMR
Abstract:
HCV is responsible for 60% of cases of chronic hepatitis and 50% of cases of cirrhosis, end-stage liver disease, and liver cancer. Four million Americans show evidence of HCV infection and the virus causes an estimated 12,000 deaths per year. No effective vaccine is available and the current treatment involving a combination of pegylated recombinant interferon-alpha-2b (rIFNa-2b) and ribavirin has toxicity issues and is nonspecific for HCV. The only therapies that directly target HCV are the recently FDA approved NS3 protease inhibitors telaprevir and boceprevir which are limited to genotype 1 and are susceptible to mutation. Therefore, there is a demand for novel approaches to treatment of HCV infection. The HCV internal ribosomal entry site (IRES) is a highly conserved 5'-untranslated region of HCV mRNA which is folded into complex secondary and tertiary conformations. It is important for initiation of viral translation and it has been shown to be important for the life cycle of the virus with domain II being essential for IRES activity. Catalytic metallodrugs offer a novel approach to inactivation of the IRES through irreversible chemistry. In addition, metallodrugs having the capacity to react with multiple RNA targets and have higher levels of specificity as a result of a double filter selection mechanism where nonspecific binding to other biomolecules does not lead to productive chemistry.
Keywords: HCV, metallodrugs
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
Due to the rise of antibiotic resistance, there is a need for discovery of new targets and development of novel drugs.1 In this study, we are targeting helix 69 (H69) of 23S ribosomal RNA (rRNA) in E. coli ribosomes with peptides as potential new antibacterial compounds.2 This RNA contains highly conserved nucleotides, in addition to three pseudouridine modifications. Helix 69 is located in the 50S subunit of the ribosome, specifically at the interface with the 30S subunit, and plays important roles in translation.3 Peptides were chosen as possible drug candidates to target modified H69 because they have reasonable stability and can be easily modified.4 Phage display was performed in order to screen for linear heptapeptides that target H69 under various conditions, such as high and low pH, differing magnesium concentrations, or in the presence of competitor RNAs.5 After four rounds of selection, the bound phage were analyzed and several consensus sequences were identified. These peptides were synthesized by using solid-phase synthesis techniques, purified by HPLC, and characterized by MALDI mass spectrometry. Binding assays with H69 revealed peptides with moderate affinity. Future studies will involve optimization of these peptides for enhanced binding and selectivity for modified H69.
References:
1. G. Taubes. The bacteria fight back. Science 2008, 321 (5887), 356.
2. J. Frank, R. K. Agrawal. A ratchet-like inter-subunit reorganization of the ribosome during translocation. Nature 2000, 406 (6793), 318.
3. A. Bakin, J. Ofengand. Four newly located pseudouridylate residues in Escherichia coli 23S Ribosomal RNA are all at the peptidyltransferase center: analysis by the application of new sequencing technique. Biochemistry 1993, 32 (37), 9754.
4. R. B. Merrifield. Solid phase peptide synthesis. I. The synthesis of a tetrapeptide, J. Am. Chem. Soc. 1963, 85, 2149.
5. G. P. Smith. Surface presentation of protein epitopes using bacteriophage expression systems. Curr. Opin. Biotechnol. 1991, 2 (5), 668.
Keywords: Helix 69, Peptides, Phage display
Abstract not available online - please check the printed booklet.
Abstract:
Nanopores are under investigation for its key role in designing rapid, low cost nucleic acid sequencing platforms. They have been utilized to detect the conformation and dynamics of polymers, including DNA and RNA. Biological nanopores are extremely reproducible with uniform channel size. The bacterial virus phi29 DNA-packaging motor is a naturally occurring biological channel for the transportation of dsDNA, and has the largest diameter among other well-studied biological channels, thus offering more space for further channel modifications and conjugation. Interestingly, this large channel, which translocates dsDNA, cannot detect single stranded nucleic acids, ssDNA or ssRNA. Here, we reengineered this motor channel to detect the real time translocation of ssDNA or RNA. One internal loop segment of the channel was removed, which results in two classes of channels, one with the same size as wild-type channel and other with cross-sectional area of 60% of the wild-type. The loop-deleted connector was able to translocate ssRNA with equal competencies and the translocation was observed to be bi-directional. This finding of size alterations in reengineered motor channels expands the potential application of the phi29 DNA-packaging motor in sensing of RNA structure and folding. We demonstrate the robust characteristics of single pore electrophysiological assays. The conductance of each class of pore is almost identical and is perfectly linear with respect to the applied voltage. Numerous transient current blockade events induced by RNA are consistent with the dimensions of the channel and nucleic acids. The connector channel is stable under a wide range of experimental conditions including high salt and pH 2-12. The robust properties of the connector nanopore made it possible to develop a simple reproducible approach for RNA quantification and characterization. The precise number of connectors in each sheet of the membrane was simply derived from the slopes of the plot of voltage against current. Such quantifications led to a reliable real time counting of nucleic acid oligomers passing through the channel. The fingerprint of RNA translocation in this system has provided a new tool for future biophysical and physicochemical characterizations of RNA.
References:
Wendell, D.; Jing, P.; Geng, J.; Subramaniam, V.; Lee, T. J.; Montemagno, C.; Guo, P. Nat. Nanotechnol. 2009, 4, 765
Geng J, Wang S, Fang H, Guo P. ACS Nano. 2013, 7, 3315
Keywords: Nanopore, Nanomotor
Abstract:
The Guanine quadruplex structure (GQS) has been of particular interest in recent years and is important to regulation of replication, transcription and translation. The general nucleotide pattern for a GQS motif is “GwLaGxLbGyLcGz”, where w, x, y, and z are ≥2 nucleotides, and loops (L) a, b, and c have lengths of ≥1 nucleotide. The unique quartet structure and folding properties of GQSs lead to unique spectroscopic features including an inverse UV melting profile at 295 nm, distinct circular dichroism (CD) features, and the recently discovered intrinsic fluorescence in DNA GQSs. We investigated the effect of increasing loop and G-stretch lengths on GQS folding in several RNA systems. We found that longer loops generally have a weaker potassium ion binding affinity (K+1/2), which is likely due to an increase in flexibility. As the length of the G-stretch increases (i.e. G2 GQS to G6 GQS), we observed a general decrease in folding cooperativity (Hill coefficient, ‘n’), leading to a broad ~5-log response range to K+ ions. We demonstrated that the observed decrease in cooperativity is due to intermediates in the GQS folding pathway, showing a clear 3-state transition in G3 and G4 GQS. Lastly, we found that RNA GQSs also exhibit the property of intrinsic fluorescence.1
The intrinsic fluorescence of GQS was further explored in DNA systems with varying loop sequence, loop length, and G-stretch length. Potassium ion titrations were performed and folding was monitored by fluorescence and CD spectroscopy. We found that longer G-stretches (i.e. G3 GQS vs G2 GQS) exhibit stronger fluorescence, even per G-stretch length. Interestingly, we also observed that the GQS with the shortest loops (1 nucleotide) have the strongest fluorescence emission intensity. Looking into the available NMR structures of dG3T and dG2A, we found that the T loops in dG3T are oriented away with respect to the G-quartets, whereas the A loops in dG2A are oriented toward and can interact with the G-quartet. This result is especially interesting as dG2A has a heptad structure, which suggests that A loops (λmax >385 nm) exhibit maximal fluorescence at longer wavelength than T loops (λmax <385 nm) due to extended conjugation. Also, the extruded orientation of the T loops in dG3T likely minimizes fluorescence quenching by loop bases and thus leads to stronger fluorescence.2
Overall, GQS folding can occur in a highly cooperative (molecular switch-like) or uncooperative (rheostat-like) mode, depending on the G-stretch length. Also, the intrinsic fluorescence of GQS is useful for nucleic acid studies and the development of label-free detection methods. These studies serve to provide a deeper understanding of structure and folding properties of GQS folding, and shed light on possible roles of GQS in cells.
References:
1. Kwok, C. K., Sherlock, M. E., and Bevilacqua, P. C. (2013) Angew. Chem. Int. Ed. 52, 683-686.
2. Kwok, C. K., Sherlock, M. E., and Bevilacqua, P. C. (2013) Biochemistry 52, 3019-3021.
Keywords: G-quadruplex, folding cooperativity, fluorescence
Abstract not available online - please check the printed booklet.
Abstract:
U12 and U6atac in minor class are functional analogs of U2 and U6 snRNAs of major class spliceosome and play a pivotal role in splicing of minor class introns. U12 snRNA binds to the branch point whereas U6atac snRNA binds to the 5' splice site after U11 interactions are destabilized. The 3' stem loop of the U6atac snRNA is considered as the guide element in minor class splicing. Various inter- and intramolecular RNA-RNA interactions are facilitated by spliceosomal proteins and other splicing factors. p65 RNA binding protein, a component of U11/U12 di-snRNP 18S complex, has two RNA binding domains. The c-terminal domain of p65 is shown to interact with terminal end of U12 stem-loop (SL) III. Our data also show that the terminal end of the U12 SL III is functionally important for in-vivo splicing. Structural and sequence similarity between the distal 3' SL of U6atac and the apical loop of U12 SL III gives rise to the notion that p65 has potential to interact with U6atac. Our data show that the distal SL of 3' U6atac and terminal loop of U12 SL III are functionally swappable, in vivo. In addition, Electrophoretic Mobility Shift Assay (EMSA) data show that p65 c-terminal domain interacts with the distal SL of 3' U6atac SL in a dose dependent manner. 3' distal U6atac SL mutagenesis analysis revealed that point mutation A99C and C100G in the loop enhanced the splicing whereas U98G did not affect the splicing. Abolishing the predicted stem by disrupting the base-pairing in the distal stem caused splicing defect. Restoration of the entire base pairing in the stem by complementary mutations couldn’t restore the WT splicing. In summary, our data show that p65 can interact with 3' SL of U6atac but its role in splicing has not yet been known.
Keywords: U12 and U6atac, minor class spliceosome, p65
Abstract:
Somatic mutations of spliceosomal proteins such as U2AF1, SF3B1, SRSF2 and PRPF8 have been identified in myeloid malignancies. These mutations are usually single heterozygous missense mutations at recurrent sites. While all of these mutations affect proteins in the common splicing pathway, their downstream consequences may be diverse and involve distinct oncogenic pathways. To identify affected genes and gain mechanistic insight into the effects of these mutations, we have analyzed the tumor transcriptomes of multiple AML patients with and without spliceosomal factor mutations. For U2AF1, we analyzed 6 tumors with U2AF1 mutations and 14 tumors that had no known splicing factor mutations. Focusing on cases of alternative exon inclusion, we identified 35 exons in 35 genes whose inclusion was altered using stringent statistical cutoffs. Of these, 8 exons were more included while 27 were more excluded in the U2AF1 mutant tumors. We examined the splice site signals flanking the alternative exons and noted a highly unusual sequence pattern adjacent to the 3’ AG dinucleotide which is the recognition site of U2AF1. The sequence at this position showed a mutually exclusive pattern upon comparing the excluded to the included exons and this pattern also differed from the consensus 3’ splice site sequence. We speculate that the RNA recognition activity of U2AF1 is altered by these mutations. We also found that these splicing changes are not seen in tumors expressing low levels of wild type U2AF1, nor are the patterns the same in similar tumors with mutations in splicing factors SF3B1, SRSF2 or PRPF8.
Keywords: Splicing factors Mutations, alternative splicing, Myeloid leukemia
Abstract:
RNA Polymerase II (RNAPII) transcribes mRNA and small non coding RNA. The C-Terminal domain (CTD) of Rbp1 plays a large role in the events and timing of transcription. The repetitive sequence YSPTSPS (26 repeats in S.cerevisiae) and its phosphorylation pattern as RNAPII moves across the gene is the main orchestrator of when proteins are recruited to the CTD and in turn controls the timing of transcription events such as termination and 3’ end processing. Messenger RNA cleavage and polyadenylation are two important processes that are coupled with RNAPII transcription termination in eukaryotes. In yeast the complexes implicated in these tasks include: cleavage factor IA (CF IA), Hrp1 (cleavage factor IB), the Nrd1-Nab3-Sen1 (NNS) complex, the cleavage and polyadenylation factor (CPF), and the nuclear exosome. Although the major subunits of each complex have been defined by previous studies, the degree of crosstalk between these complexes and RNAPII has not been determined. By utilizing affinity purifications coupled with mass spectrometry (MudPIT) and quantitative proteomics analysis, we will determine the degree of involvement RNAPII plays in 3’ end processing complex formation, the recruitment dynamics of subunits involved, , and any post translation modifications that may occur. We will present evidence showing that many of the 3’ end processing complexes interact with RNAPII. However, we observe limited co-purification of subunits from different 3’end processing complexes suggesting that RNAPII and/or the nascent RNA likely provide the framework for 3’ end processing complex crosstalk. In addition, we have identified a number of novel candidates associated with specific mRNA processing complexes that we plan to characterize further.
Keywords: RNA Polymerase II, 3 end processing, mRNA
Abstract:
In recent years, there has been increasing interest in the function of numerous post-transcriptional modifications of tRNA bases and sugars that are known to occur in all three domains of life. While roles for some modified nucleotides found near the tRNA anticodon in translation are relatively well-established, the biological function for modifications found in the remaining tRNA body is far less well-understood. Some modifications occurring in the tRNA core affect overall stability of the tRNA, and thus, loss of specific tRNA modifications in this region can lead to degradation via the rapid tRNA decay pathway. In addition, cells exposed to oxidative stress or growth arrest gain additional modifications on certain tRNAs. Thus modification of tRNA can be regulated in cells as a way to ensure overall quality and function of the tRNA pool, but the specific consequences of alternative tRNA modification patterns remain to be fully investigated.
In this work, we show that the yeast m1G9 methyltransferase, Trm10, displays the ability to modify additional tRNA substrates, both in vitro and in vivo, beyond the set of tRNA species that are normally modified in wild-type S. cerevisiae. We hypothesize that this expanded mode of substrate specificity is advantageous in that it allows Trm10 to modify non-cognate tRNAs in cells under stress, possibly preserving the structural integrity of the tRNA. We analyzed the modification status of the 5'-end of tRNAs exposed to two different stress conditions using primer extension. We show that when cells are either exposed to 5-fluourouracil or lack certain tRNA modification enzymes, additional primer extension stops corresponding to known tRNA modifications, including m1G9, are observed. These data support the hypothesis that the modification status of tRNAs is more dynamic than previously understood and opens the door to investigation of the physiological function of alternative tRNA modification patterns in cells.
Keywords: tRNA modification, Trm10, stress
Abstract:
DEAD-box RNA helicases use ATP to bind and remodel RNA and RNP complexes. In vitro, most DEAD-box proteins display little sequence specificity, but in the cell many of these enzymes are thought to target specific RNAs. Although most eukaryotic DEAD-box proteins have been implicated in defined cellular processes, it is often not known which RNAs are targeted and where targeted RNAs are bound. The absence of this critical in¬formation greatly complicates the under¬standing of biological functions of DEAD-box proteins on a molecular scale. Here, we define binding sites in RNA targets of the DEAD-box helicase Ded1p from Saccharomyces cere¬visae. We combined in vivo crosslinking of genomically encoded, histidine-biotin (HB) affinity tagged Ded1p with protein purification under denaturing con¬ditions, followed by cDNA library genesis from crosslinked RNAs and Next Generation Sequencing. Ded1p has been implicated in various cellular processes, including translation initiation and ribosome biogenesis. Accordingly, we find that Ded1p binds (pre-) ribosomal RNA and a large cross-section of expressed mRNAs. The number of mRNA sequence reads correlates broadly with the mRNA expression level, consistent with a role of Ded1p as a general translation initiation factor. Bindings sites of Ded1p are distributed along the entire length of mRNAs, and many are found in the ORF. The binding sites do not have an apparent sequence signature, however a major binding site of Ded1p is seen slightly downstream of the initiation codon.
Keywords: RNA, CLIP, DEAD-box
Abstract not available online - please check the printed booklet.
Abstract:
Human lysyl-tRNA synthetase (LysRS) is critical for the replication of Human Immunodeficiency Virus 1 (HIV-1), as it directs the selective packaging of host tRNALys3, the primer for HIV-1 reverse transcription, into HIV-1 virions. Like several other aminoacyl-tRNA synthetases (aaRSs), LysRS has been demonstrated to perform a number of non-canonical functions beyond its role in aminoacylating or “charging” amino acids onto their cognate tRNAs. In most of these cases, LysRS becomes activated to perform non-canonical functions through post-translation modifications (PTMs), which cause it to dissociate from the high molecular weight multi-synthetase complex (MSC), where it exists as a homotetramer in complex with 8 other aaRSs and 3 scaffold proteins. In addition, three forms of LysRS are produced from the same gene: the cytoplasmic isoform, an immature pre-mitochondrial isoform, and a processed mitochondrial isoform. The exact isoform(s) packaged into HIV-1 virions has been a point of debate. We are using liquid chromatography-coupled tandem mass spectrometry to identify whether the PTM profile of human LysRS is altered upon HIV-1 infection, as well as to establish the specific isoform(s) of LysRS that is/are packaged into HIV-1 virions. To date, we have identified both cytoplasmic and pre-mitochondrial isoforms within HIV virions, and we will employ a targeted MS approach to identify the presence or absence of the mature mitochondrial species. While preliminary data suggest that LysRS is post-translationally modified, changes in the PTM profile remain to be elucidated. LysRS proteolysis is being optimized to provide better sequence coverage and SILAC studies are planned to probe changes in LysRS modifications in HIV-1 infected cells.
Keywords: HIV, Aminoacyl-tRNA Synthetase, Mass Spectrometry
Abstract:
Recently named one of the top five emerging pathogens by the CDC, Enterovirus 71 is a positive single stranded RNA virus belonging to Picornaviridae. Due to the lack of a 5’ cap, EV71 utilizes an Internal Ribosome Entry Site (IRES) to regulate and promote the translation of viral proteins required for viral replication. A key ITAF involved in this Protein:RNA interaction is human hnRNP A1 which upon infection translocates from the nucleus to the cytoplasm to interact with Stem Loop II (SLII) and Stem Loop VI (SLVI). Little is known on how hnRNP A1 interacts with these two independently folding stem loops; we posit that RNA structure, as well as sequence, influences this Protein:RNA interaction. To characterize this interaction a series of bioinformatics, biophysical and virological techniques were utilized. We show that: (1) a high degree of secondary and sequence conservation is present within the IRES; (2) hnRNP A1 interacts biphasically with SLII at two distinct sites, a lower bulge with a conserved UAG motif and an upper hairpin containing a conserved CCA nucleotide motif and, (3) mutation of the UAG binding site in vivo leads to a significant reduction in viral translation and replication. To better elucidate this interaction a preliminary high resolution model of SLII was determined. Delineation of this interaction may pave the way for novel anti-viral therapies and treatments.
Keywords: IRES, hnRNP A1, ProteinRNA Interactions
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
Introns are transcribed DNA segments which are excised from the primary transcript by splicing together the flanking exons. Major eukaryotic intron classes include the GU/AG and AU/AC spliceosomal introns, self-splicing group I and group II introns, tRNA introns, and the hac1 (yeast)/xbp1 (Drosophila/human) gene intron class which plays a role in the unfolded protein response. Drosophila rnp-4f is a nuclear gene containing nine classical spliceosomal introns which encodes an evolutionarily conserved protein functioning as a chaperone enabling association between U4- and U6-snRNAs during spliceosome assembly. During an extensive screen of polyadenylated EST clones derived from D. melanogaster developmental stages, we unexpectedly found transcripts containing an identically spliced 58-nt segment from some 0-4 h embryo and also pupal clones. Excision of this intron causes a frame-shift which completely changes the encoded C-terminus and results in a long unique 83 amino acid sequence tract. This intron does not conform to any of the major intron classes, and does not arise from a second rnp-4f gene. We have identified several interesting correlations between the Drosophila xbp1 gene intron and that of the novel rnp-4f intron: (1) both produce rare mRNA transcripts, the xbp1 class being highly expressed during stress; (2) intron splicing in both creates a frame-shift which results in a long unique C-terminal amino acid sequence; (3) both are flanked by a short direct repeat (C-U-G-C-N-G-C in xbp1 and C-C-C-A-C-C-N-C in rnp-4f); (4) both excision sites occur 3-nt from a stem-loop at the 5’-splice junction; and (5) the intronic secondary structure in both brings the 5’- and 3’-ends into close proximity for subsequent ligation. Intron excision in hac1/xbp1 mRNAs is via Ire-1 endonuclease (Drosophila CG45830), while the putative rnp-4f endonuclease remains to be discovered. We have made an antibody specific to the long unique C-terminal amino acid sequence resulting from splicing by this new intron class, which should enable locating sites within fly embryos where this event occurs and to eventually determine its functional significance.
Keywords: rnp-4f gene, non-spliceosomal intron, novel new intron class
Abstract not available online - please check the printed booklet.
Abstract:
Mapping and sequencing of individual transfer ribonucleic acids (tRNAs) can prove to be very difficult, requiring rigorous sample preparation prior to analysis. Previously our group has presented a simplified method for the comparative analysis of RNA digests (CARD).1,2 In the CARD approach two complete sets of digestion products from total tRNA are compared using the enzymatic incorporation of 16O/18O isotopic labels. With this approach we are able to rapidly screen total tRNAs from gene deletion mutants or comparatively sequence total tRNA from two related bacterial organisms. However, data analysis can be challenging due to convoluted mass spectra arising from the natural 13C and 15N isotopes present in tRNA samples. Here, we demonstrate that culturing in 12C-enriched/13C-depleted media significantly reduces the isotope patterns that must be interpreted during the CARD experiment. Improvements in data quality enable one to use existing software analysis tools to reduce data processing steps, and these improvements enable one to obtain more biologically relevant information from the analysis.
References:
1. Siwei Li and Patrick A. Limbach (2012). Method for comparative analysis of ribonucleic acids using isotope labeling and mass spectrometry. Analytical chemistry, 84 8607–8613.
2. Siwei Li and Patrick A. Limbach (2013). Mass spectrometry sequencing of transfer ribonucleic acids by the comparative analysis of RNA digests (CARD) approach. The Analyst, 138 1386–1394.
Keywords: Mass spectrometry, Isotope enrichment depletion, Modified RNA
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
The tRNAHisguanylyltransferase (Thg1) is part of a family of enzymes found in all three domains of life, members of which catalyze an unprecedented 3’—5’ nucleotide addition reaction. Structural similarities between canonical DNA/RNA polymerases and eukaryotic Thg1 suggest unexpected connections between these enzyme activities. Recent structure and transient kinetic analysis of human Thg1 for incorporation of the guanine residue to the -1 position (G-1) of tRNAHis reveals several mechanistic features of 3’-5’ addition. However, many mechanistic questions, particularly related to the ability of Thg1 to bind to its tRNA substrates, remain unsolved. Since tRNA is a relatively large molecule, similar in overall molecular weight to the mass of a single Thg1 monomer, andThg1 has been observed to exist as a tetramer, we hypothesize that the multimeric form of the enzyme is an important feature allowing recognition of the large tRNA substrate. For this reason, we chose to investigate several highly conserved residues that are predicted to be involved in Thg1 multimerization. Previously, variants of human Thg1 believed to disrupt the dimer-dimer interface of the enzyme based on their positions in the crystal structure have been characterized in vitro. The purpose of this project was to test the function of these particular variants in vivo in yeast. This was accomplished using a yeast complementation assay to test whether the variant was able to support growth in the absence of the wild type THG1 gene. The in vivo results were found to largely mirror the previous in vitro characterization, supporting the predicted role of these particular residues in holding the dimer-dimer interface together.
Keywords: Thg1, tRNAHis
Abstract not available online - please check the printed booklet.
Abstract:
tRNAs are major components of the cell’s protein synthesis machinery. In addition to this essential role in gene expression, they also contribute to other diverse functions including protein degradation, apoptosis, cellular response to stress, and tumorigenesis. tRNAs are transcribed in the nucleus. After the removal of the 5’ and 3’ ends and the addition of CCA and some modifications, tRNAs are exported to the cytoplasm where they complete their biogenesis and fulfill their functions. In both yeast and vertebrate cells, the subcellular movement of tRNAs involves the initial export of tRNAs from the nucleus to the cytoplasm, retrograde nuclear import of cytoplasmic tRNAs, and re-export of the imported tRNAs back to the cytoplasm. Although tRNAs have been studied for decades, some major players in tRNA metabolism and subcellular movement remain unknown. For example, there is an unknown nuclear export pathway for intron-containing tRNAs in yeast. The overall aim of this study is to identify and characterize all the missing gene products involved in tRNA biology, using yeast, S. cerevisiae, as a model organism. Our strategy is to conduct a genome-wide assessment of the impact of every gene upon tRNAs utilizing the yeast deletion and temperature-sensitive (ts) collections. To conduct this screen in a timely fashion, we developed a rapid and sensitive method for genome-wide analysis of small RNAs from strains in the mutant collections (Wu et al., Yeast). To date, all the 4848 mutants in the deletion collection have been analyzed; candidates that affect tRNA biology have been identified and verified. For example, there are several novel candidates involved in tRNA biosynthesis steps including 5' and 3' end processing of the initial transcript and removal of introns in the cytoplasm. This study will uncover important factors that function in tRNA metabolism and intracellular trafficking, which will contribute to a better understanding of the complexity of tRNA biology.
Keywords: tRNA, genome-wide analysis
Abstract:
Cell regulatory circuits integrate diverse and conflicting environmental cues to generate appropriate condition-dependent responses. Here, we elucidate the components and mechanisms driving a protein-directed RNA switch in the 3'UTR of vascular endothelial growth factor (VEGF)-A [1]. We describe a novel HILDA (hypoxia-inducible hnRNP L–DRBP76–hnRNP A2/B1) complex that coordinates a three-element RNA switch, enabling VEGFA mRNA translation during combined hypoxia and inflammation. In addition to binding the CA-rich element, heterogeneous nuclear ribonucleoprotein (hnRNP) L regulates switch assembly and function. hnRNP L undergoes two condition-dependent post-translational modifications: IFN-γ induces prolyl hydroxylation and von Hippel-Lindau (VHL)-mediated proteasomal degradation; whereas, hypoxia stimulates hnRNP L phosphorylation at Tyr359, inducing binding to hnRNP A2/B1 which stabilizes the protein. Phospho-hnRNP L recruits DRBP76 (double-stranded RNA binding protein 76) to the 3'UTR where it binds an adjacent AU-rich stem-loop element, “flipping” the RNA switch by disrupting the GAIT (Gamma-interferon Activated Inhibitor of Translation) element, preventing GAIT complex binding, and driving robust VEGFA mRNA translation. We exploited a genome-wide analysis of hypoxia-induced RNA switches that suggest a widespread posttranscriptional regulon that might contribute to tumorigenesis. We show that halofuginone (HF), a derivative from a Chinese herbal-based medicine, can enhance GAIT-mediated translational silencing activity and reverse GAIT-dependent RNA switches. Also, we demonstrate that CA-rich miR-574-3p acts as a decoy of hnRNP L, which binds CA-rich regions of mRNAs, and prevents HILDA binding of target mRNAs and reduces translation of the entire pool of HILDA-directed RNA switch transcripts. Our studies reveal novel molecular mechanisms, biological significance, and therapeutic potential of stress-responsive HILDA-directed RNA switches.
References:
1. Peng Yao, Alka A. Potdar, Partho Sarothi Ray, Sandeepa M. Eswarappa, Andrew C. Flagg, Belinda Willard, and Paul L. Fox. The HILDA complex coordinates a conditional switch in the 3’-untranslated region of the VEGFA mRNA. Plos Biology 2013(11): e1001635
Keywords: RNA switch, VEGF-A, miRNA
Abstract not available online - please check the printed booklet.
Abstract:
The results of the ENCODE project and other large scale transcriptome analyses suggest that while over 75% of the human genome is transcribed into RNA, ORFs and their associated UTRs occupy only 2% of the genome. It is estimated that the human genome contains over 70,000 large non-protein coding transcripts (lncRNAs); however, many aspects of the biology and function of this novel class of cellular regulators are almost completely unknown.
We have analyzed a ~2700 nucleotide long nuclear transcript that is spliced and polyadenylated but has no protein-coding capacity. Interestingly, we found that the expression of the lncRNA is induced when cells are exposed to stressful conditions in several different cell types, including cells of mesodermal and ectodermal origin. We analyzed the expression of factors that are involved in cellular stress response and apoptosis in cells that either overexpressed the lncRNA or in which the RNA was knocked down using shRNA-mediated strategies. After exposure to stress, the knockdown cells exhibited an impaired cellular stress response and a much higher death rate compared to controls, while the overexpression cells showed an elevated level of stress response and a cellular stress-resistant phenotype. Analysis of the stress response factors indicated that some but not all heat shock proteins had a higher basal level in the overexpression cells even in the absence of stress and thus, the elevated expression of the lncRNA seemed to “pre-condition” the cells for tolerance of stress. Interestingly, in the knockdown cells, the induction of heat shock proteins after stress was both delayed and reduced in magnitude. We made a series of truncation mutations in the lncRNA and could show that deletion of part of the RNA close to its 5' end abolished its ability to impart stress resistance in transfected cells, while the other mutants did not have this effect. Together, these data indicate that this lncRNA plays an important role in regulation of the stress response and cellular survival under stressful conditions and demonstrates the power of lncRNAs as a novel class of regulatory molecules in higher eukaryotes.
Keywords: large non coding RNA, lncRNA, Stress
Abstract:
A major problem in health care today is the emergence of multi-drug resistance in bacterial pathogens. In efforts to find ways to evade these resistance mechanisms, numerous derivatives of the aminoglycoside (AG) tobramycin (TOB) were made and tested for biological activity against a broad spectrum of bacteria (1). Here, these TOB derivatives, with various substitutions at the 6’’-position of ring III, are further characterized with respect to their mode of action. We tested the ability of each to inhibit (i) growth of E. coli strain ∆7 prrn (WT), (ii) growth of aminoglycoside-resistant E. coli ∆7 prrn (A1408G), (iii) translocation in WT ribosomes, and (iv) translocation in A1408G ribosomes. Based on the data obtained, the variants fall into distinct functional classes. Those in class A inhibit growth and translocation in A1408-dependent manner, like the parental TOB, suggesting that act by binding the primary h44 site. These compounds are substituted with similarly-sized groups (tert-butyl, benzyl, p-methyl benzyl, cyclohexyl) at the 6’’-position. Class B variants inhibit translocation in an A1408-dependent manner but fail to inhibit growth, suggesting that they target h44 but bind with low affinity or accumulate to lower levels in the cell. Class C variants inhibit both translocation and growth, but only the former activity is A1408-dependent. Presumably, these compounds target h44 and something else in the cell. Class D variants inhibit translocation and growth, and neither activity depends on A1408. These compounds may target another ribosomal site and potentially another cellular component. Class E variants inhibit growth in an A1408-independent manner and fail to inhibit translocation, indicating that they target something other than the ribosome. A common feature of most of the class C, D, and E compounds is a linear alkyl chain extending from the 6’’ position. A subset of these compounds cause cell lysis (1), implicating the membrane as a cellular target in these cases. Together these data provide insight into h44 binding and translocation inhibition by AG antibiotics.
References:
1. I. M. Herzog, K. D. Green, Y. Berkov-Zrihen, M. Feldman, R. R. Vidavski, A. Eldar-Boock, R. Satchi-Fainaro, A. Eldar, S. Garneau-Tsodikova, and M. Fridman, Angew. Chem. Int. Ed. 2012, 51, 5652.
Keywords: Aminoglycoside, h44, ribosome
Abstract:
Synthesis of long RNAs by RNA polymerases (RNAP) requires accessory factors that help RNAP to overcome numerous roadblocks along the template. RfaH, the best characterized regulator capable of switching the elongating RNAP into a processive state, belongs to the only universally conserved family of transcription factors. RfaH is recruited to the transcription elongation complex via direct interactions with bases of a 12-nt ops element in the non-template DNA strand exposed on the surface of RNAP. The individual roles of these 12 nucleotides remain unclear: while some nucleotides may directly interact with RfaH, others could exert their effects through interaction with RNAP. Initial molecular modeling suggested that only the upstream 6 ops nucleotides can interact with RfaH, but in vivo lux reporter assays demonstrated that substitutions in the downstream half of the ops element have equally strong effect on gene expression. The latter substitutions may interfere with RNAP pausing at ops T11 position, which was hypothesized to be essential for RfaH recruitment. Recent structural studies suggested that pockets on RNAP may contribute to base-specific recognition of the template and non-template DNA strands during transcription. In this work, we probed the roles of ops nucleotides in RNAP pausing and RfaH recruitment.
Keywords: RfaH recruitment, ops element, base specific recruitment
