RRM
2012 Rustbelt RNA Meeting
RRM
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Poster abstracts
Abstract:
Transposable elements (TEs) are mobile fragments of DNA that occupy large fractions of eukaryotic genomes. Their ability to move makes them potent mutagens, and eukaryotes have evolved elaborate mechanisms of TE repression that utilize small RNAs (sRNAs) to direct heterochromatic epigenetic modifications to TE sequences. This mode of TE silencing is conserved from plants to mammals and has well-documented effects on the regulation of TE-neighboring genes in cis. My research has uncovered a novel mechanism of TE regulation of genes in trans, whereby an Arabidopsis thaliana TE represses a non-TE-neighboring gene via a TE sRNA, resulting in altered regulation of the cellular stress response. A class of Arabidopsis sRNAs accumulate only under conditions of genome-wide transcriptional reactivation of TEs. We provide direct evidence that one of these sRNAs, siRNA854, post-transcriptionally regulates the non-TE gene, UBP1b. We are the first to demonstrate that the epigenetic regulation of a TE can lead to the regulation of a gene in trans, mediated by a TE sRNA. This work blurs the lines between TE-derived and gene-regulating sRNAs, a distinction that is prevalent in the current literature.
Based on this research, we hypothesized that a genome-wide mechanism of gene regulation exists in which TE-derived sRNAs act to alter global gene regulation under conditions of transcriptionally active TEs. To test this hypothesis, I paired sRNA deep sequencing with microarray analysis of wild-type and mutant plants, which lose global epigenetic regulation of TEs, in order to find candidate TE-derived sRNAs that putatively target non-TE genes. The relationship between TE sRNAs and putative target candidates was further analyzed to obtain a small list of candidates on which to focus future experiments. We believe that post-transcriptional regulation of genes by TE sRNAs may contribute to the changes in gene expression and phenotype observed in plants and animals during viral infection and/or progressive loss of heterochromatin, as new TE and viral small RNAs and new unexplained phenotypes are abundant under these conditions.
Keywords: small RNA, transposable elements, post-transcriptional gene regulation
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
In many Bacilli, the undecameric (11-mer) toroidal protein TRAP acts as a tryptophan sensor, becoming allosterically activated by tryptophan to bind to specific mRNAs. TRAP binding to these mRNAs favors the formation of alternate secondary structure elements, which abort the production of nascent transcripts or block translation of completed mRNAs. A second protein, the trimeric Anti-TRAP (AT), is expressed under tryptophan starvation conditions and prevents the TRAP/RNA interaction by occluding the RNA binding site of TRAP.
Solution-state studies of TRAP-AT complexes via analytical ultracentrifugation (AUC) and Small-Angle X-Ray Scattering (SAXS) reveal that complexes assembled at low AT:TRAP ratios (i.e., 1-2) are oligomeric and heterogeneous, likely consisting of multiple AT trimers and TRAP 11-mers. Interestingly, as the ratio of AT to TRAP is increased, dimensions of these complexes dramatically decrease.
To determine the structural configurations adopted under various conditions by TRAP-AT complexes, we have developed software that aims to identify a minimal ensemble of structures consistent with a range of structural data, including SAXS, AUC, and FRET. The software utilizes a genetic algorithm capable of determining the best-fitting ensemble from thousands of candidate structural conformers. Using this approach, we analyzed SAXS solution-state titration data from 4 to 8 AT3:TRAP11 ratios and observe that the experimental data are reliably fit when two complex configurations are present: TRAP in which all five binding sites are occupied by AT trimers, and TRAP bound to only three AT trimers. This result is consistent with isothermal titration calorimetry data that reveal 2-3 high affinity, and 2-3 low affinity binding modes.
This global analysis method represents a powerful approach by which to study the highly variable structural configurations, and has already provided important insight into the TRAP-AT interaction.
Keywords: SAXS, Regulation, TRAP
Abstract:
Germline mutations in neurofibromatosis type 1 (NF1) result in NF1 disease, which presents with multiple symptoms including an increased risk of cardiovascular diseases. Studies using several different mouse models further established the important role of Nf1 in the cardiovascular system. Neurofibromin, the protein encoded by Nf1, functions as a Ras-GAP (GTPase-activating protein). It promotes the conversion of active Ras-GTP to inactive Ras-GDP via its GAP-related domain (GRD). One of its alternatively expressed exons, exon 23a, lies in the middle of the GRD of neurofibromin. The impact of expression of this exon on Ras-GAP activity of endogenous Nf1 is poorly understood. Our lab has generated two ES cell lines through gene targeting that express exon 23a at either 0% or 100% and demonstrated that expression of exon 23a controls the active Ras output in ES cells with low exon 23a expression correlating with high active Ras level. To investigate the role of Nf1 exon 23a expression in the cardiovascular system, the two ES cell lines were differentiated into beating cardiomyocytes. The primary beating cardiomyocytes and the wild-type ES cell derived cardiomyocytes show a similar level of Nf1 exon 23a expression at 70%. The mutated cells maintain the expected pattern of exon 23a expression throughout the process of differentiation. They also show the expected differences in active Ras-levels and phosphorylation levels of downstream targets of the Ras signaling pathway. Surprisingly, the cardiomyocytes differentiated from these cells exhibit statistically significant differences in both the number of beating embryoid bodies (EBs) and the beating frequency. The cardiomyocytes with no expression of exon 23a beat faster and have higher percentage of beating EBs than their counterparts with 100% expression of exon 23a. In addition, real time RT-PCR analysis examining the transcript levels of progenitor and differentiated cardiomyocyte markers during a time course of differentiation suggests that the cells with no expression of exon 23a differentiate faster and these cardiomyocytes show the highest expression of cardiomyocyte specific genes. These studies indicate that proper splicing of NF1 exon 23a is important for cardiomyocyte differentiation by maintaining the appropriate Ras-GTP levels.
Keywords: alternative splicing, NF1
Abstract not available online - please check the printed booklet.
Abstract:
During the retroviral lifecycle, specific host cell tRNAs are used as primers for reverse transcriptase (RT). In the case of HIV-1 and Rous sarcoma virus (RSV), tRNALys,3 and tRNATrp, respectively, serve this role. These tRNAs are selectively packaged along with their cognate lysyl- and tryptophanyl-tRNA synthetases (LysRS and TrpRS). Retroviral assembly is tightly regulated through interactions involving the Gag protein, which directs the assembly process, viral genomic RNA, and cellular factors. HIV-1 Gag is required for packaging of LysRS, and the RT domain of Gag-Pol is implicated in selective tRNALys packaging. The RT region of RSV Gag-Pol is crucial for tRNATrp recruitment, but the interactions that dictate TrpRS packaging are unknown. In this work, using antibodies against chicken TrpRS we confirmed the packaging of TrpRS into RSV particles. Moreover, our preliminary immunofluorescence confocal imaging studies suggested that a subpopulation of TrpRS was co-localized with Gag and the viral RNA in the nucleus. We hypothesize that RSV Gag-TrpRS interactions may be initiated in the nucleus, and studies to probe both Gag-TrpRS and genome-TrpRS co-localization are underway.
In HIV-1, the 3’-18 nt of tRNALys,3 anneal to a complementary primer binding site (PBS) located near the 5’ end of the genome. A region upstream of the PBS strongly resembles the tRNALys anticodon loop, which is the major binding/recognition element of the synthetase. This “tRNA‐Like Element” (TLE), binds LysRS with a similar affinity as tRNALys. Moreover, cell-based assays suggest that LysRS-TLE interaction facilitates placement of tRNALys onto the PBS and contributes to viral infectivity. By analogy to this work, a putative tRNATrp anticodon domain mimic has been identified in the RSV genome. Fluorescence anisotropy binding assays suggest a short, 60 nt, and longer, 419 nt, segment of the genome containing the TLE region, are able to bind to and compete with tRNATrp as a substrate for TrpRS. Future studies will test the role of TrpRS/genomic RNA interaction on tRNATrp priming and viral infectivity.
Keywords: RSV, TrpRS, TLE
Abstract not available online - please check the printed booklet.
Abstract:
Decapping, exonuclease trimming and endonuclease cleavage generate RNAs with a 5’ monophosphate end. In mammalian cells these products are degraded by Xrn1, they can be stored uncapped or they can undergo recapping by the cytoplasmic capping enzyme complex. Capturing sites of decapping and cleavage, collectively known as the degradome, provides us clues to the events occurring in the lifetime of the transcriptome. We have developed an efficient deep sequencing strategy to specifically amplify and sequence uncapped 5’ ends. With some modifications, this technique can also be adopted for also detecting capped 5’ ends. The method uses an RNA adapter to tag the 5’ end of monophosphorylated RNAs followed by cDNA synthesis using a 5’ monophosphorylated N6 random primer yielding 5’ monophosphorylated cDNA fragments. cDNAs with sequence complementary to the 5' RNA adapter undergo a second-strand synthesis using an RNA adapter specific DNA oligo and a thermophilic DNA polymerase. The double-stranded cDNAs are 3' adenylated and ligated to adapters followed by a low cycle PCR amplification for deep sequencing. Phosphorylation of the cDNA strand selectively allows for ligation of the DNA adapter. The PCR products (150-400 bp) are gel eluted and subjected to qPCR analysis to verify the specific amplification of 5' ends of the known uncapped RNA molecules such as MAPK1, EXOSC1, 28S and 18S rRNAs. The method is highly quantitative, sensitive and precise in capturing known amounts of spiked 5’ monophosphorylated luciferase RNA down to attograms levels with a remarkably low intra-sample variation (Coefficient of variation=0.07, <1 is considered low variance). With barcoding, this method can be easily adopted for multiplexing several samples and is currently being used to capture the RNA recapping sites in mammalian cells.
Supported by grant R01 GM084177 from the National Institute of General Medical Sciences of the National Institutes of Health.
References:
Mukherjee C, Patil DP, Kennedy BA, Bakthavachalu B, Bundschuh R, Schoenberg
DR. Identification of Cytoplasmic Capping Targets Reveals a Role for Cap Homeostasis in Translation and mRNA Stability. Cell Rep. 2012 Aug 21. [Epub ahead of print] PubMed PMID: 22921400.
Affymetrix ENCODE Transcriptome Project; Cold Spring Harbor Laboratory ENCODE Transcriptome Project. Post-transcriptional processing generates a diversity of 5'-modified long and short RNAs. Nature. 2009 Feb 19;457(7232):1028-32. Epub 2009 Jan 25. PubMed PMID: 19169241; PubMed Central PMCID: PMC2719882.
Karginov FV, Cheloufi S, Chong MM, Stark A, Smith AD, Hannon GJ. Diverse
endonucleolytic cleavage sites in the mammalian transcriptome depend upon
microRNAs, Drosha, and additional nucleases. Mol Cell. 2010 Jun 25;38(6):781-8.
PubMed PMID: 20620951; PubMed Central PMCID: PMC2914474.
Keywords: Uncapped RNA, deep sequencing, Recapping
Abstract:
Ribonuclease P (RNaseP) is an enzyme essential in all forms of life, and is composed of both RNA and protein subunits. Across the domains of life, the singular RNA moiety of this ribonucleoprotein (RNP) complex has been shown to be catalytic on its own in vitro, although the protein subunits are required for in vivo activity. The number of protein subunits varies from 1 in Bacteria to 9 or 10 in Eukarya. We have used nuclear magnetic resonance (NMR) to analyze a protein-protein interaction and used molecular modeling to model the protein-RNA interactions between various subunits of RNase P from the hyperthermophilic Archaeon Pyrococcus furiosus (Pfu). Pfu RNase P possesses five protein subunits, four of which form two binary pairs RPP21-RPP29 and RPP30-Pop5. Dynamic light scattering (DLS) indicates that the RPP30-Pop5 dimer forms a heterotetramer in solution. NMR chemical shift perturbations map the interaction surface for the complex between RPP30 and Pop5, in agreement with the crystallographic studies of a homologous pair from another Archaeon. Finally, structural homology involving the Pfu RPR and Pop5 with the bacterial RPR and C5 protein from Thermotoga maritima (Tma) enables the RPP30-Pop5 complex to be positioned onto a model of the Pfu RPR. These results continue to add insight into the interactions involved in RNP formation and will inform future efforts to elucidate assembly and structure of the whole RNase P complex.
Keywords: RNase P, Protein-Protein interaction, Protein-RNA interaction
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:
Prolyl-tRNA synthetase (ProRS) is a class II synthetase that aminoacylates proline onto cognate tRNAPro. ProRSs commonly misactivate alanine and cysteine, which must be edited to avoid incorporation into the translating protein. The so-called “triple-sieve” editing mechanism used by ProRS involves a cis editing domain on ProRS that hydrolyzes Ala-tRNAPro and the single-domain YbaK protein, which edits mischarged Cys-tRNAPro in trans. Previous fluorescence anisotropy studies suggested that YbaK and ProRS interact in vitro and the binding affinity is increased in the presence of tRNAPro. Additionally, chemical cross-linking studies support the formation of a ProRS/YbaK/tRNAPro ternary complex in vitro. Recent work examining these interactions in vivo using a split-GFP reassembly assay (B.R. So, PhD Thesis, 2010) and an independent cross-linking approach (L. Nie and T. Magliery, unpublished), support YbaK/ProRS complex formation. Although these studies indicate that YbaK, ProRS, and tRNAPro interact as a ternary complex, there is currently no high-resolution, three-dimensional structure of this interaction. The present work is focused on defining the interaction between tRNAPro and YbaK at the molecular level. Two dimensional 15N-H NMR spectra of 15N-enriched YbaK have been obtained in the absence and presence of tRNAPro. Preliminary data indicate that a subset of peaks shift or change in intensity upon tRNAPro binding and assignment of these peaks is currently underway. In addition to the NMR studies, footprinting and cross-linking studies are being conducted to define the tRNAPro/YbaK interaction interface. These studies in combination with computational docking will yield the first three-dimensional model of this trans editing complex.
References:
An, S. and K. Musier-Forsyth, Cys-tRNAPro editing by Haemophilus influenzae YbaK via a novel synthetase/YbaK/tRNA ternary complex. J Biol Chem, 2005. 280(41): p. 34465-72
Keywords: tRNA, aminoacyl tRNA synthetase
Abstract not available online - please check the printed booklet.
Abstract:
Helix 69 (H69) is located within the large subunit of the bacterial ribosome and has been implicated to have a role in protein synthesis. Previously, the phage display technique provided the peptide sequence NQVANHQ, which was shown to bind to H69 with moderate binding affinity. In this study, modifications of this peptide were produced using solid-phase synthetic approaches. This technique offers an efficient way of synthesizing heptamers with various modifications. Fourteen peptides were synthesized for the initial stage of this project. Binding studies using the electrospray ionization mass spectrometry (ESI-MS) technique yielded a dissociation constant for each peptide – RNA complex. These studies provide information regarding which functional groups on the peptide are necessary for binding to H69. Using ESI-MS, competition reactions between modified peptides and the parent sequence NQVANHQ were performed to further study binding efficiency. A combination of these peptide modifications could provide an optimized binder with greater binding affinity to the target RNA which can be further tested to identify potent inhibitors of protein synthesis of the bacterial ribosome.
Keywords: peptide, helix 69, ribosome
Abstract:
DNA and RNA assemblies rely on crossovers to form three way junctions (3WJ) or four way junctions (4WJ) that enhance nanostructures beyond simple helical duplexes. Here we show that direct incorporation of branches within the backbone provide simple means to enhance DNA and RNA assemblies. Copper (I) promoted azide- alkyne cycloaddition (CuAAC), commonly referred to as click chemistry is a specific reaction that can covalently link an azide and an alkyne under simple conditions, creating a triazole linkage. Standard phosphoramide chemistry allows incorporation of azide and alkyne groups with nucleic acid sequences of interest, to generate a stable backbone branched structure after a click reaction. Melting curves of these branched sequences show that the linked sequences behave as separate duplexes when hybridized to their complementary sequences. However the branched sequences can act as a single unit creating an efficient scaffold for a fluorescent nanotag that gives rise to a multi chromophore system. Intercalating dyes within the branched duplexes add up to a higher extinction co-efficient of donor dyes which can excite an acceptor dye of interest, via efficient Foster resonance energy transfer for multiplex assays. Such brighter nanotags are of significance for biological imaging applications.
Keywords: Backbone branched nucleic acids, Click chemistry, Foster resonance energy transfer
Abstract:
Typical in vitro transcription assays use templates that are made of linear, double-stranded DNA. In this study we used novel templates that possessed a double-stranded promoter for T7 RNA polymerase recruitment, but the remainder of each DNA construct contained three different downstream variations. The control template utilized a linear, completely double-stranded DNA template. This was compared to two templates of the same length where one template strand was self-complementary and formed a hairpin (snapback), whereas the other strand possessed no self-complementary and remained single-stranded. All three templates produced RNA that was similar in length by denaturing polyacrylamide gel analysis. The kinetic data for each template could be fit by relatively simple Michaelis-Menten parameters. We found variation among the templates utilized at different temperatures. Results will be shown.
Keywords: T7 RNA Polymerase, kinetics
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
In every round of translation elongation, EF-G catalyzes translocation, the movement of tRNAs (and paired codons) to their adjacent binding sites in the ribosome. Previous kinetic studies have shown that the rate of tRNA-mRNA movement is limited by a conformational change in the ribosome termed unlocking. While structural studies offer some clues as to what unlocking might entail, the molecular basis of this conformational change remains an open question. In this study, the contribution of intersubunit bridges to the energy barrier of translocation was systematically investigated. Unlike those targeting B2a and B3, mutations that disrupt bridges B1a, B4, B7a, and B8 each increased the maximal rate of both forward (EF-G dependent) and reverse (spontaneous) translocation. As bridge B1a is predicted to constrain 30S head movement and B4, B7a, and B8 are predicted to constrain intersubunit rotation, these data provide evidence that formation of the unlocked (transition) state involves both 30S head movement and intersubunit rotation.
Keywords: ribosome, translocation, inter-subunit bridge
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
Recently we developed CARD, a method for the Comparative Analysis of RNA Digests. By differential labeling of two samples using 16O and 18O, any RNA digests that are identical in the two samples will appear as doublets in mass spectral analysis separated by 2 Da. More significantly, differences between the two samples are represented by singlet peaks. For many applications, the differences between two samples will be minor meaning that much of the mass spectral data will be doublets with only a few singlets. The challenge in data analysis is to rapidly identify and characterize these singlets. To address this challenge, we introduce an Excel interface program based on Visual Basic for Application (VBA) for analysis of CARD data. Liquid chromatography-mass spectrometry (LC-MS) test data were obtained from previous studies. The automated processing steps in this program drastically reduce the need for manual processing of LC-MS data by applying filters to remove hundreds of doublets and noise in the mass spectra. Three example CARD datasets have been used in program development, and results from the application of this program to these datasets will be presented. Further algorithm development and optimization is on-going.
Keywords: Comparative analysis of RNA, LC-MS, Algorithm
Abstract not available online - please check the printed booklet.
Abstract:
In Xenopus, maternal stored mRNAs with a short poly (A) tail (less than 40nt) are repressed by a deadenylase (PARN) before oocyte maturation (1). However, those mRNAs are activated by a cytoplasmic poly (A) polymerase (GLD-2) associating with the cytoplasmic polyadenylation complex and function in oocyte maturation (1).
In plants, cytoplasmic polyadenylation has not been described. However, it has been reported that seeds contain a sizeable population of stored mRNA (2). In addition, germination and its completion are less sensitive to de novo transcription inhibitors than to poly (A) polymerase inhibitors (3,4). Together, these considerations suggest that stored RNA with a short poly (A) tail (stored spRNA) may have a function in seed germination upon reactivation by cytoplasmic polyadenylation. To further explore this, mRNA polyadenylation has been studied through the course of germination using a combination of transcriptional inhibitors and a modified RNA-seq strategy (5). Using this approach, several putative stored spRNAs have been identified. These stored spRNAs candidates are being further characterized using S1 nuclease protection- and qRT-PCR-assays. Progress along these lines will be discussed.
References:
1. Villalba A, Coll O, Gebauer F. 2011.Curr Opin Genet Dev 4:452-7.
2. Harris, B., and L. Dure, 3rd. 1978. Biochemistry 17:3250-6.
3. Tao, K. L., and A. A. Khan. 1976. Plant Physiol 58:769-772.
4. Datta, K., L. Marsh, and A. Marcus. 1983. Plant Physiol 72:394-397.
5. Wu, X., Liu, M., Downie, B., Liang, C., Ji, G., Li, Q. Q., and Hunt, A. G. 2011. Proc Natl Acad Sci U S A 108:12533-12538.
Keywords: cytoplasmic polyadenylation, stored RNA, seed germination
Abstract not available online - please check the printed booklet.
Abstract:
The RNA-activated protein kinase, PKR is an RNA-binding protein and an essential sensor in the innate immune response. Upon binding of RNA, PKR can dimerize and undergo autophosphorylation, followed by phosphorylating eIF2alpha, which leads to termination of translation and eventually apoptosis. PKR has a well- characterized role in recognizing viral RNA and initiating the immune response in order to rid the virus. PKR is known to bind long stretches (>33 bp) of double-stranded RNA (dsRNA) non-sequence specifically, but recently has been shown to be more permissive, binding other functional, biological and non-conventional RNAs, as well as some proteins. The hypothesis is that PKR binds novel RNAs in vivo and a subset of them are oxidatively damaged, which leads to the known upregulation of PKR in age-related diseases. Many biological RNAs are prone to oxidative damage and this leads to diseases. Because of the known upregulation of PKR in cells of patients affected with age-related diseases, specifically Alzheimer’s disease (AD) and Parkinson’s disease (PD), there is potential that PKR interacts with RNAs that lead to these diseases. Cells affected with these diseases have stressed environments, partially due to reactive oxidative species produced by some of the disease-related proteins. There is an overall relation between innate immunity (PKR), oxidative stress and age-related diseases and my goal is to develop a mechanistic and molecular understanding of these phenomena. An investigation of PKR activation by a wide variety of RNAs including those related to AD is being performed under normal conditions and after the RNAs have been oxidatively damaged (by multiple methods) and I will present my recent results.
Keywords: Alzheimers Disease, PKR, RNA binding protein
Abstract:
The MDM2 oncogene encodes a protein that negatively regulates p53 by targeting it for proteasome-mediated degradation. Through the induction of DNA damage and in cancer, MDM2 is alternatively spliced into several isoforms. MDM2-ALT1, comprised of exons 3 and 12, is observed in over 85% of rhabdomyosarcomas, is generated in cells in response to genotoxic stress and has also been shown to accelerate tumorigenesis in a mouse model, all of which indicate a role in cancer.
In order to study the alternative splicing of MDM2 we have developed an in vitro splicing system using MDM2 minigenes and normal and cisplatin-treated HeLa nuclear extracts. The MDM2 3-11-12 minigene predominantly excludes exon 11 under UV and cisplatin treatment both in vivo and in vitro. Using ESEfinder 3.0 we identified putative binding sites for splicing regulators SC35 and SF2/ASF in exon 11 of the MDM2 minigene and performed a series of mutations in their predicted binding sites.
Our in vitro data shows that disrupting the SC35 sites in exon 11 leads to exclusion of exon 11 and disrupting the SF2/ASF site promotes the inclusion of exon 11. However, in MCF7 and HeLa cells transfected with MDM2 3-11-12 minigenes, both SC35 and SF2/ASF site mutants promote the inclusion of exon 11 under normal, UV and cisplatin treatment. We confirmed the affinity of these regulatory proteins through RNA oligonucleotide pull downs using the wild-type and mutant sequences of each binding site.
Finally, we designed antisense oligonucleotides to target these splicing regulatory sites endogenously. All ASOs blocked the formation of MDM2-ALT1 and restored full-length MDM2 in MCF7 cells upon cisplatin treatment. Taken together, this indicates that these sites are important for regulating the damage-induced alternative splicing of MDM2 and offers the potential of anti-sense-based therapy as a means of corrective splicing in cancer.
Keywords: MDM2, Alternative Splicing, SF2ASF
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
Ribonucleoprotein (RNP) complexes perform crucial roles in diverse cellular processes including DNA replication, repair, RNA transcription and maturation, and protein synthesis. The ability to accurately determine the absolute quantity of proteins within such complexes provides useful information relating to complex stoichiometry and biogenesis. Here, mass spectrometry based-approaches are being used to characterize and obtain absolute quantitative information of proteins associated with RNPs. By virtue of parallel data acquisition, alternating scans of low energy collision-induced dissociation (CID) and high-energy CID during liquid chromatography-mass spectrometry analyses reveal both protein identification and quantitation in a single experiment. The low energy CID mode is used to obtain accurate mass measurements of precursor ions and intensity data for quantitation, while the high-energy CID mode generates peptide fragmentation of all precursor ions for database searching and subsequent protein identification. Because the information obtained in these experiments is related to the number of peptides identified per protein (within the complex), optimization of experimental conditions is necessary to provide an unbiased analysis of all low and high molecular weight components. The approach was developed using E. coli wild-type ribosomes and then applied to ribosome assembly particles that accumulate as a result of perturbation. Ribosomal proteins and extra-ribosomal protein factors associated with these particles were identified. In addition, absolute quantitative information of proteins provided insights on the degree of heterogeneity of these ribonucleoprotein particles. This MS-based platform enables the rapid characterization of RNA-protein complexes including ribosome assembly particles resulting from perturbations, stress conditions and deletion strains of proteins or assembly factors.
Keywords: ribosomal proteins, ribonucleoprotein complexes, mass spectrometry
Abstract:
In recent years the complexity of the eukaryotic transcriptome has become a subject of intense curiosity as well as debate. It is now well established in eukaryotic organisms from yeast to humans, that RNA polymerase II (pol II) transcribes hundreds to thousands of long non-coding RNAs (lncRNAs). While our knowledge of the mechanisms and scope of lncRNA-mediated regulation is growing, our understanding of how lncRNAs themselves are regulated is still quite limited. Here we demonstrate that lncRNA abundance is regulated by decapping-dependent decay. As pol II transcripts messenger RNAs (mRNAs) and lncRNAs bear distinctive features at their extremities (i.e. a 5’ cap and 3’ poly(A)tail). A highly conserved pathway for the destruction of mRNAs requires removal of the cap structure (i.e. decapping) which then leaves the body of the transcript susceptible to 5’ to 3’ exonucleolytic digestion. Decapping represents a critical control point in regulating mRNA expression because it commits the transcript body to destruction. While the role of decapping in controlling mRNA levels is well documented, the contribution of decapping in modulating the levels and function of other capped RNAs has been largely unexplored. In this study we document that decapping influences the expression of >100 lncRNAs in S. cerevisiae through a novel decapping-dependent pathway that occurs independent all known mRNA decapping regulators. We find that decapping-sensitive lncRNAs are often expressed proximal to inducible genes. Using galactose inducible genes as a model for lncRNA-mediated regulation, we show that upon stimulation clearance of the lncRNA in the nucleus by decapping-dependent decay is required for rapid and robust gene activation. Failure to destabilize this lncRNA, which is known to exert repressive histone modifications, results in perpetuation of a repressive chromatin state that contributes to reduced plasticity of gene activation. We propose that decapping-dependent decay serves a vital role in regulating lncRNA-mediated epigenetic events at inducible genes.
Keywords: lncRNA, RNA decapping, RNA decay
Abstract:
The noncoding RNAs, including siRNA, miRNA, ribozyme, and riboswitch, have been found participating in the regulation of cell life cycle and their applications in medical and pharmaceutical areas have become routine practice. Although the folding, degradation, and intracellular half-life of RNA are interested in many studies, current methods to observe intracellular RNAs are extremely challenging. The common method to measure RNA half-life in vivo is the use of radioactive markers or fluorescence RNA labeling. The challenge is, after the degradation of RNA the isotope or the fluorescence is still present in the cell, thus the signals are not a true indication of the presence of the RNA in the cell. Here we report a method to monitor RNA degradation in real time in living cells using fluorogenic RNA aptamer in combination with RNA nanotechnology. The RNA aptamer that binds Malachite Green (MG), the ribozyme that cleaves the hepatitis virus genome, and a siRNA for firefly luciferase were all integrated into a 3WJ derived from bacteriophage phi29 packaging RNA (pRNA) to generate RNA nanoparticles. MG is an organic dye which is not fluorogenic by itself, but it starts emitting fluorescent light when it binds to the RNA aptamer. The binding only happens when the RNA is folded in the correct conformation. Therefore, the MG aptamer fluorescence can be used as a measure of the degradation and folding of the RNA nanoparticles using epifluorescence microscopy and fluorescence spectroscopy method without lysing the cells. The half-life of the electroporated RNA nanoparticle containing MG aptamer was found to be 4.3 hours after electroporation into cells.
References:
1. Reif R, Haque F, Guo P. Fluorogenic RNA Nanoparticles for Monitoring RNA Folding and Degradation in Real Time in Living Cells. In press.
2. Shu D, Shu Y, Haque F, Abdelmawla S, Guo P. Thermodynamically stable RNA three-way junction as a platform for constructing multifunctional nanoparticles for delivery of therapeutics. Nature Nanotech. 2011; 6(10):658-67.
3. Haque F, Shu D, Shu Y, Shlyakhtenko LS, Rychahou PG, Evers BM, Guo P. Ultrastable synergistic tetravalent RNA nanoparticles for targeting to cancers. Nano Today. 2012. 7: 245—257
4. Guo P. The emerging field of RNA nanotechnology. Nature Nanotech. 2010; 5:833–842.
Keywords: RNA, fluorescence tag, aptamer
Abstract not available online - please check the printed booklet.
Abstract:
Telomerase is a ribonucleoprotein enzyme that is typically required for sustained cell proliferation. Although both telomerase activity and the telomerase catalytic protein component, TbTERT, have been identified in the eukaryotic pathogen Trypanosoma brucei, the RNA molecule that dictates telomere synthesis has remained unknown. Here we identify the RNA component of Trypanosma brucei telomerase, TbTR, and provide both in vivo and in vitro evidences for TbTR’s native folding and activity. We show that TbTR is processed through trans-splicing, is a capped transcript that interacts and co-purifies with TbTERT in vivo. Deletion of TbTR caused progressive shortening of telomeres at a rate of 3–5 bp/PD, which can be rescued by ectopic expression of a wild-type allele of TbTR in an apparent dose-dependent manner. Remarkably, introduction of mutations in the TbTR template domain resulted in corresponding mutant telomere sequences, demonstrating that telomere synthesis in T. brucei is dependent on TbTR. We also propose a secondary structure model for TbTR based on phylogenetic analysis, chemical and enzymatic mapping experiments, thus defining TbTR domains that may have important functional implications in telomere synthesis. Identification and characterization of TbTR not only provides important insights for investigation of T. brucei telomere functions, which have been shown to play important roles in T. brucei pathogenesis, but also offers T. brucei as an attractive model system for studying telomerase biology in pathogenic protozoa and for comparative analysis of telomerase function with higher eukaryotes.
Keywords: telomerase, noncoding RNA, Trypanosoma brucei
Abstract not available online - please check the printed booklet.
Abstract:
P5ab is a RNA hairpin, i.e. an A-form double helix, which is the basic unit of RNA structure. It is part of the catalytic core in the Tetrahymena thermophila ribozyme. Since its identification by Cech and coworkers, this RNA hairpin has been studied by X-ray crystallography, biochemical methods, temperature jump and single-molecule assays by several groups over the decades [1-4]. It has been previously characterized as a two-state folder in single-molecule studies [3, 4]. Here using single-molecule force spectroscopy with resolutions of sub-nanometer and millisecond, we first identify a fast intermediate state I (rate constant ~ 400 /sec) along the folding pathway of P5ab, suggesting a more complex folding route than initially thought. Then to better characterize state I, a mutant m1 is designed by replacing two G-U wobbles with G-C pairs in the hairpin stem, resulting in a more stable state I (rate constant ~30 /sec), which now makes the hairpin (un)fold hierarchically in two discrete steps. Based on the m1 sequence, a mutant m2 aimed at creating a new intermediate I2 is achieved by insertion of an A-A bulge between state I and the tetraloop. It turns out such mutation also changes the folding cooperativity of the hairpin, which now unfolds all the way to the intermediate I2, bypassing state I. Next we demonstrate in mutant m3 how to stabilize another intermediate I1 by both base insertions and replacements. Finally we reconstructe the free energy landscape profiles from the extension probability under constant force followed by the deconvolution procedure. We present, to our knowledge for the first time, the full sequence-dependent folding landscapes of the RNAs, which demonstrate how single-base mutations in the sequence can drastically alter the folding kinetics, pathways and cooperativity. Such detailed information will be crucial for understanding mechanisms by which molecular motors make genetic decisions based on RNA folding kinetics.
References:
1. J. H. Cate, A. R. Gooding, E. Podell, K. Zhou, B. L. Golden, C. E. Kundrot, T. R. Cech, J. A. Doudna, Crystal structure of a group I ribozyme domain: principles of RNA packing. Science 273, 1678 (1996).
2. S. K. Silverman, T. R. Cech, Energetics and cooperativity of tertiary hydrogen bonds in RNA structure. Biochemistry 38, 8691-8702 (1999).
3. J. Liphardt, B. Onoa, S. B. Smith, I. Tinoco Jr., C. Bustamante, Reversible unfolding of single RNA molecules by mechanical force. Science 292, 733 (2001).
4. J. Wen, M. Manosas, P. T. X. Li, S. B. Smith, C. Bustamante, F. Ritort, I. Tinoco, Jr., Force unfolding kinetics of RNA using optical tweezers. I. Effects of experimental variables on measured results. Biophys. J. 92, 2996 (2007).
Keywords: P5ab, optical tweezers, free energy landscape
Abstract:
Aminoacyl-tRNA synethetases activate specific amino acids and transfer them to cognate tRNA isoacceptors. These enzymes are error prone due to misactivation of smaller or isosteric amino acids. In the case of prolyl-tRNA synthetase (ProRS), in addition to activating cognate Pro, Ala and Cys are readily misacylated. Thus, editing mechanisms have evolved to ensure fidelity of Pro codon translation. In many bacterial systems, a triple-sieve editing mechanism is employed, which consists of the ProRS active site that discriminates amino acids based largely on volume and size, the ProRS editing domain (INS) that hydrolyzes Ala-tRNAPro and also functions via size exclusion, and a free-standing INS homolog called YbaK, which clears Cys-tRNAPro via unique sulfhydryl side-chain chemistry. The gram-negative purple non-sulfur bacterium Rhodopseudomonas palustris (Rp) encodes a ProRS containing a truncated (non-editing) INS domain in addition to two single-domain homologs YbaK and ProX. Similar to Ec ProRS, Rp ProRS activates cognate Pro and misactivates noncognate Ala and Cys. Rp ProRS shows pre-transfer editing activity against Ala that is comparable to that of Ec ProRS and in the presence of tRNA, the synthetase robustly mischarges Ala onto tRNAPro. Therefore, despite the lack of a full INS domain and the relatively robust pre-transfer editing activity of Rp ProRS, we hypothesize that editing of Ala-tRNAPro is required. Additionally, efficient activation of Cys coupled with minimal Cys pre-transfer editing and good mischarging activity implies that editing of Cys-tRNAPro is essential. Here, we show that the mini-INS lacks hydrolytic activity, as expected, but appears to be critical for Rp ProRS structural stability. Also, Rp YbaK possesses robust Cys-tRNAPro editing activity. The function of ProX has not been reported for any species. We demonstrate here that Rp ProX inhibits mischarging of Ala on to tRNAPro in the presence of ProRS. Thus, in Rp an alternative triple sieve editing mechanism appears to exist in which the catalytic domain acts as the coarse sieve, and the single-domain proteins ProX and YbaK act as fine sieves to prevent formation of misacylated Ala-tRNAPro and Cys-tRNAPro, respectively.
Keywords: aminoacyl tRNA synthetase, post-transfer editing, tRNA
Abstract not available online - please check the printed booklet.
Abstract:
Through transcriptome profiling of the Schizosaccharomyces pombe genome, we have identified a natural antisense transcript at the alcohol dehydrogenase 1 (adh1) locus that is induced in response to zinc-limitation. This antisense transcript (adh1AS) shows an expression pattern reciprocal to that of its mRNA partner, adh1. We have found that expression of the adh1AS transcript in low zinc is necessary to repress expression of adh1. Our data also shows that the adh1AS transcript is regulated at multiple levels. It is regulated at the transcriptional level by changes in intracellular zinc. In addition to this transcriptional regulation, when the adh1AS transcript is placed under a heterologous promoter, it preferentially accumulates under zinc-limiting conditions. We have found that this second level of regulation is dependent on expression of adh1, but is independent of RNAi. Together, these results suggest a novel mechanism of strand-specific RNA accumulation in response to changes in nutrient levels, and provide insight in determining how antisense transcripts can be regulated at multiple levels.
Keywords: yeast, antisense transcription, zinc
Abstract:
Multi-drug resistance among bacterial pathogens can be mediated by a number of mechanisms, including efflux pumps. One such pump in Bacillus spp. is ykkCD, a heterodimer of the small multidrug resistance (SMR) family consisting of C and D subunits. Previous studies suggest that the expression of the gene is controlled by a putative riboswitch and that the antibiotic tetracycline binds to the riboswitch in vitro. Additional studies have shown that two derivatives of tetracycline also bind to the putative riboswitch. These findings now need to be confirmed in vivo. This study will explore the effects that tetracycline and its commercially available derivatives—doxycycline, anhydrotetracycline, oxytetracycline, and minocycline—have on the expression levels of the ykkCD gene in Bacillus subtilis. The level of ykkCD gene expression will be quantified using two different methods: (1) cellular ykkCD mRNA levels will be determined by quantitative RT-PCR and (2) ykkCD protein levels will be estimated using a ykkCD RNA-beta-galactosidase reporter gene construct. Together these results will shed light on the antibiotic specificity of the ykkCD RNA in vivo.
Keywords: Multi-drug resistance, Riboswitch, qRT-PCR
Abstract:
Enterovirus 71 (EV71), a positive single stranded RNA virus in the picornaviridae family, is one of the two major etiological agents involved in hand, foot and mouth disease. The lack of a 5’ m7G cap results in the utilization of a highly structured six stem loop IRES and various host IRES trans-acting factors (ITAFs) during translational initiation (1). A key ITAF implicated in EV71 IRES activity is heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) which is believed to interact with Stem Loop II (SLII) in the EV71 IRES, making it likely that SLII is critical to IRES dependent activity. To examine the nature of hnRNPA1:SLII interactions, a bioinformatics, NMR and Isothermal Calorimetric (ITC) based approach was utilized. To elucidate characteristic SLII sequence and secondary structure similarities between different EV71 strains, complete genome alignment and SLII secondary structure prediction occurred. All predicted EV71 SLII secondary structures presented a phylogenetically conserved secondary structure. This high degree of secondary and primary sequence conservation in the bulge and hairpin regions make it likely that these regions are critical to hnRNPA1:SLII interactions. 2D NOESY analysis coupled to secondary structure prediction confirmed the presence of the upper six nucleotide hairpin and the lower five nucleotide bulge in SLII. To explore the hnRNPA1 binding nature of these regions, ITC studies on a wildtype SLII and a hairpin only mutant were performed. Wildtype ITC data suggests a propensity for hnRNPA1 to bind to SLII in a sequential manner involving two unique binding sites, i.e. the lower bulge and upper hairpin. To examine this likelihood, a hairpin only mutant was designed to examine binding exclusive to the hairpin; an additional in vivo viral activity assay was also utilized. The high degree of secondary structure conservation coupled to observed hnRNPA1 binding patterns independent of the bulge make it likely that SLII could act as a novel EV71 therapeutic site.
References:
1. Lin, J., et. al. (2009) hnRNP A1 Interacts with the 5’ Untranslated Regions of Enterovirus 71 and Sindbis Virus RNA and Is Required for Viral Replication. Journal of Virology 83: 6106-6114.
Keywords: EV71, hnRNPA1
Abstract:
Ebola virus is a negative sense RNA virus that can cause viral hemorrhagic fever. The nucleocapsid structure includes the RNA and several viral proteins, with the nucleoprotein (NP) directly interacting with the RNA. To characterize the mechanism involved in this RNA-protein interaction we are establishing a source of pure, soluble NP. Using protein secondary structure prediction software (Poodle, PredictProtein, and PSIPRED) we identified the C-terminus of NP as likely to be intrinsically disordered and thus a potential culprit for the aggregation seen during previous NP purification attempts. We therefore constructed plasmids which coded for the N-terminus of NP with various C-terminal truncations for bacterial overexpression. Truncating NP at amino acid 356 resulted in purification of soluble protein, even after cleavage of the maltose binding protein used to stabilize NP during the expression and purification process. Experiments to study the interactions of NP with the viral RNA, as well as efforts to determine the structure of NP, are being pursued.
References:
1. E. T. W. Bowen, G. Lloyd, W. J. Harris, et al., Lancet 1, 571 (1977).
2. M. A. Bwaka et al., J. Infect. Dis. 179 (suppl 1), S1 (1999).
3. L. H. Elliott, M. P. Kiley, J. B. McCormick, Virology 147, 169 (Nov, 1985).
4. H. Feldmann, H. D. Klenk, A. Sanchez, Arch. Virol. Suppl. 7, 81 (1993).
5. M. P. Kiley, R. L. Regnery, K. M. Johnson, J Gen Virol 49, 333 (Aug, 1980).
Keywords: Ebolavirus, Nucleoprotein, RNA
Abstract not available online - please check the printed booklet.
Abstract:
Actinomycetes Nocardiopsis are ecologically versatile, and have been isolated from a variety of habitats ranging from human blood to shallow water sediments. We have identified Nocadiopsis alba strain BE74 as a persistent member of the gut microbiota of honeybees in Southern Ohio. The same species was also isolated from beehives in Thailand. BE74 shows inhibitory activity against honeybee pathogens Paenibacillus larvae and other indigenous Bacillus bacteria in beehives. We sequenced the whole genome of BE74 with next-generation sequencing to provide a foundation for further studies on this bacterial genus. Through genome mining, we identified a cluster of three genes (albABC) encoding enzymes for biosynthesizing albonoursin, an antibiotic secondary metabolite. Albonoursin is a cyclodipeptide composed of phenylalanine and leucine. AlbC is the key biosynthetic enzyme catalyzing the formation of the two peptide bonds of cyclodipeptide ring structure by using aminoacyl-tRNAs as substrates. In a sense, the charged tRNAs were hijacked by AlbC from protein synthesis of primary metabolism to supply the biosynthesis of a secondary metabolite. To study the cellular effects of the cyclodipeptides and the regulation of albABC gene expression, we first constructed a ΔalbABC mutant by homologous recombination using a cosmid library and the PCR targeting system. The Nocardiopsis mutant, confirmed by Southern blotting analysis, lost the production of the cyclodipeptide. The production was restored after complementing the mutant with the albABC genes on a replicative plasmid. We then introduced a β-Glucuronidase gene into the mutant chromosome to monitor transcriptional activity of the promoter of the albABC genes. To further investigate the relationship between aminoacyl-tRNAs and the biosynthesis of secondary metabolites in bacteria, we plan to utilize the whole-genome sequenced Nocardiospis alba as a model to carry out transcriptomic, metabolomic and tRNAnomic studies.
References:
(1)JianJun Qiao, Lei Chen, Yongli Li, Jiangxin Wang, Weiwen Zhang, and Shawn Chen. Whole-Genome Sequence of Nocardiopsis alba Strain ATCC BAA-2165 Associated with Honeybees. J Bacteriol.2012 (accepted )
(2)Patil PB, Zeng Y, Coursey T, Houston P, Miller I, Chen S.(2010). Isolation and characterization of a Nocardiopsis sp. from honeybee guts. FEMS microbiology letters, 312(2), 110-8. doi:10.1111/j.1574-6968.2010.02104.x
Keywords: aminoacyl-tRNAs, cyclodipeptides, Nocardiopsis alba
Abstract:
Recent advances in pharmacogenomics indicate the importance of RNA in the modulation of pharmacogenetic therapeutic outcomes. RNA provides an effective and critical insight into the understanding of pharmacodynamics of several prominent drug targets in various human disorders. Genome-wide association studies of SNPs, mRNA expression, and trait mapping, reveal the role of regulatory polymorphisms in several mRNA processes such as splicing, transport, stability and translation. Despite the significant role that genes often play in affecting the course of a disease and the treatment outcome, the nature and extent of genetic variability have been inadequately explored. The finding that has come out of the human genome project is the importance of the non-coding region that encodes miRNAs that are predicted to regulate one-third of the human genome, can be used to help predict drug response. Cumulating evidence now demonstrates that non-coding RNA levels in the cell can be a principal determinant of drug sensitivity or resistance, and may serve as biomarkers to assess the associated risk factor.
Our approach is to integrate RNA biology techniques to study the global transcriptome changes of pharmacological relevance in physiologically relevant human autopsy brain tissues by using Next-generation sequencing. Through 'Expression Genetics in Drug Therapy' program at the Department of Pharmacology of the Ohio State University, we aim to fill an important gap, through a systematic study of gene regulation of drug metabolizing enzymes and receptors. This would eventually pave a way into personalized medicine and therapy.
Supported by NIH grant U01 GM092655
Keywords: Transcriptome, pharmacogenomics, non-coding RNA
Abstract:
To generate the correct sequence for coding proteins, the pre-mRNA has to remove its non-coding intronic sequences and join the coding exons through the process of splicing. During the process of splicing, the introns are removed as a special lariat structure where the 5'-end of the RNA is tied back to the 2'-OH of an internal adenosine nucleotide. Recent reports indicate that some of these lariat introns after getting linearized by the lariat debranching enzyme (Dbr) can form active pre-microRNA. Therefore we sought to synthesize siRNAs (21-22 mer) where only the guide or the anti-sense strand of the siRNA is in a lariat form. As the 5'-end of these lariats are not free, they are more stable towards nuclease degradation. We used a 2'-photoprotected adenosine phosphoramidite at the branch point of the growing RNA strand to make the lariat. After the synthesis of the linear strand, we generated a 5'-phosphoramidte in the solid phase using literature adapted method. Then selective unmasking of the 2'-OH of the branch point adenosine using UV light followed by coupling of the 5'-phosphoramidite and the deprotected 2'-OH resulted in the lariat RNAs. These synthetic lariats are substrates for the lariat debranching enzyme (Dbr) in vitro. We tested the activity of these lariat RNAs in Drosophila S2 cells using a dual luciferase reporter system. Our positive results suggest that synthetic mini lariat RNAs may represent a significant new class of Drosha and Dicer independent RNA silencing agent.
References:
1. Ruby, J.G., Jan, C.H., and Bartel, D.P. Intronic microRNA precursors that bypass Drosha processing. Nature 448: 83-86 (2007).
Keywords: Lariat Intron, Debranching enzyme(Dbr), siRNA
Abstract:
Backbone branching occurs in RNA sequences during splicing. In splicing, the protein coding regions (exons) are joined and the introns are removed as lariats to generate the correct mRNA. The lariat structure includes a 2’,5’-branch. Thus, access to backbone branched RNA (bbRNA) is important to investigate splicing, debranching and other regulatory processes. There have been significant efforts to obtain bbRNAs yet, the ability to obtain natural and the modified sequences have been elusive. We report a simple strategy for the solid-phase synthesis (SPS) of bbRNAs using a photolabile protecting group. Synthesis of bbRNAs starts with incorporation of 2'-photoprotected amidite in the stem as the branching point. Photodeprotection followed by incorporation of reverse amidites provide the bbRNAs. However, to include modifications in the 2'- branch reverse amidites with the desired modification are required. As these are not available, we used a branching amidite with 3'-photoprotecting group which enable us to avoid modified reverse amidites. We observed 2'- to 3'- branch migration (and vice versa) during the photodeprotection step. That could be resolved by the selective removal of cyanoethyl group to generate phospodiester linkage (before the photodeprotection step and branch synthesis). Debranching of these modified bbRNAs by Dbr is presented. This synthetic approach will provide an insight into the role of bbRNAs in debranching, splicing and other regulatory processes.
Keywords: splicing, debranching enzyme, backbonebranched RNA, lariat
Abstract:
The 5´ untranslated region (5´UTR) of the human immunodeficiency virus type 1 (HIV-1) RNA genome (vRNA) controls virus replication via its rich secondary structure. For example, the dimer initiation sequence (DIS) and Psi hairpins control the coordinated process of vRNA dimerization and packaging. While the secondary structure of the 5´UTR is well annotated, a model for the tertiary structure of the entire 5´UTR remains elusive. The overall goal of this project is to elucidate the global fold of various RNA constructs derived from the 5´UTR of the vRNA using small angle X-ray scattering (SAXS). The 5´UTR is hypothesized to be a riboswitch that exists in a monomeric state in its role in viral protein translation and in a dimeric state that promotes packaging and virus assembly. Initial structural studies are being performed on monomeric 5´UTR constructs in which the DIS has been mutated to prevent dimerization. SAXS requires homogeneous sample preparation, which we accomplished through optimization of construct design and RNA refolding, and purification via size-exclusion chromatography (SEC). We show that by replacing divalent Mg2+ cations with monovalent K+ cations during folding of the 5´UTR constructs, sample homogeneity was increased. Additionally, by removing Mg2+ from the SEC/storage buffer, the 5´ UTR constructs remain stably monomeric at 4˚C after SEC purification. These optimization steps have yielded 5´UTR RNA constructs that are amenable to SAXS analysis and the results of initial SAXS studies will be presented. This work represents an important step towards building a low-resolution model of the entire 5´UTR in the absence and presence of interacting partners.
Keywords: HIV-1, SAXS, 5UTR
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 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 and transient kinetic analysis of human Thg1 reveals several mechanistic features of 3’-5’ addition, including a striking similarity between Thg1 and canonical DNA/RNA polymerases. 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, 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 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. Future research will include the in vitro characterization of new residues of interest that will hopefully shed light on the functional residues in the enzyme.
Keywords: Thg1
Abstract not available online - please check the printed booklet.
Abstract:
U12 and U6atac snRNAs play a key role in the minor class splicing. U12 binds to the branch point, whereas regions, whereas U6atac binds to the 5' splice site after the U11 interactions are destabilized. The 3' stem loop of the U6atac RNA 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 complex, has two RNA binding domains. Previous data show that the c-terminal domain of p65 interacts 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 give rise 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 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 predicting 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 the plasticity of RNA-RNA base-pairing interactions in U12-dependent spliceosome.
Keywords: U12 snRNA, U6atac snRNA, Splicing
Abstract:
Recognition of viral RNA by mammalian cells is critical for the activation of the innate immune system. Viral RNA is recognized by several pathogen recognition receptors, including RIG-I, an ATP-dependent RNA helicase. RIG-I binds to the termini of RNA duplexes, but it is not clear which exact conformational elements in the RNA trigger activation of RIG-I. To address this question, we examined RNA binding, ATPase activity, and oligomerization of RIG-I for a series of defined model substrates in vitro. We find that RIG-I forms oligomers on duplexes with more than 16 bp. While RIG-I binds to RNA duplexes with and without blunt ends, ATPase activity is only activated by RNA duplexes containing a blunt end. Finally, we observe that dissociation of RIG-I from RNA is enhanced by ATP. Collectively, our data indicate that the length of the duplex and exact blunt ends of RNA duplexes are critical for RIG-I activation. Our observations further indicate that RIG-I forms oligomers on duplex RNAs, but only the terminal protomer hydrolyzes ATP.
Keywords: RIG-I, ATPase, oligomerization
Abstract not available online - please check the printed booklet.
Abstract:
Pseudouridine synthases isomerize uridine (U) to its C-glycoside isomer, pseudouridine (Ψ) in RNA. Based on sequence and structural similarity, six families of Ψ synthases have been identified, which are typically named after their first cloned member: TruA, TruB, RluA, RsuA, TruD, and Pus10 [1]. Pus10, a member of the most recently identified Ψ synthase family isomerizes U55 in archaeal tRNAs [2] and also transforms the adjacent U54 to Ψ54 [3].
This work examines the activity of Methanococcus jannaschii Pus10 towards both U55 and U54 in stem-loop RNA substrates. The radiolabeling/TLC method used in a previous study [2-3] has been replaced by a less cumbersome HPLC method. Pus10 clearly isomerizes U55 more efficiently than U54. The temperature dependence of the reaction rates and preliminary kinetic data are also presented.
References:
1. Mueller, E. G., and Ferré-D'Amaré, A. R. (2009) Pseudouridine Formation, the Most Common Transglycosylation in RNA, In DNA and RNA Modification Enzymes: Structure, Mechanism, Function and Evolution (Grosjean, H., Ed.).
2. Roovers, M., Hale, C., Tricot, C., Terns, M. P., Terns, R. M., Grosjean, H., and Droogmans, L. (2006) Formation of the conserved pseudouridine at position 55 in archaeal tRNA, Nucleic Acids Res 34, 4293-4301.
3. Gurha, P., and Gupta, R. (2008) Archaeal Pus10 proteins can produce both pseudouridine 54 and 55 in tRNA, RNA 14, 2521-2527.
Keywords: Pseudouridine synthases , Pus10, HPLC method
Abstract not available online - please check the printed booklet.
Abstract:
Alternative splicing is an essential step in the HIV life cycle. It is primarily regulated by host alternative splicing factors that bind different splice sites within the viral RNA genome, which function to repress or enhance splicing activity. Splice site A7 (ssA7, ~175nts), which is phylogenetically conserved, is composed of 3 stem loops: SL1, SL2, and SL3. These stem loops contain the ISS (SL1), ESE3 (SL2), and ESS3 (SL3) splicing regulatory elements (SRE). The host proteins hnRNP A1 and ASF bind to the SRE of ssA7 to repress or enhance splicing activities, respectively. Knowledge of the structural characteristics of ssA7 will serve to better understand the mechanisms of interaction between ssA7 and hnRNP A1/ ASF. We seek to determine a high resolution NMR structure of ssA7. Due to the inherent size limitation of NMR, the spectral analysis of large RNA constructs is cumbersome. To circumvent this challenge, we have decided to segmentally incorporate an isotope labeled SL3 fragment to an unlabeled SL12 fragment. Conditions were first optimized using synthetic DNA splints to form a well-defined ligation competent complex (LCC), as analyzed by non-denaturing PAGE. We found that both the length and the amount of splint are critical variables. However, the ligation yields were still very low. We hypothesized the problem might be the inefficient 5’-priming of SL3 with GMP. To explore this hypothesis, we developed a facile 1D 31P-NMR method to detect the relative amount of 5’-GMP SL3/5’-GTP SL3. The 1D 31P NMR spectrum of 5’-GMP gives rise to a well-resolved downfield phosphorus signal, which allows us to track the efficiency of 5’-GMP priming. In sum, we found that the length and amount of DNA splint affects LCC yields and we present a facile 1D 31P-NMR method for detecting 5’-GMP incorporation. This approach should prove useful to others who wish to prepare segmental labeled RNA contructs.
Keywords: HIV, Splice Site A7, ESS3
Abstract:
Alternative splicing plays a critical role in the lifecycle of the human immunodeficiency virus, transforming one strand of pre-mRNA into over forty different mRNA transcripts. Various combinations of acceptor and donor splice sites function to accomplish such variability, with the aid of host proteins to influence the assembly of the spliceosome. One such host protein is the heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), known to abrogate spliceosome assembly by binding to highly conserved acceptor splice sites A2, A3, and A7. Splice site A7 is composed of three stem loops, intron splicing silencer (SLISS), exon splicing enhancer 3 (SLESE3), and exon splicing silencer 3, (SLESS3), all known to interact with hnRNP A1 through a yet undetermined mechanism. As a way to further define the protein-RNA binding mechanism, a mutational study was conducted to evaluate the role of nucleotides that compose the SLESS3 hairpin. For this study, the UP1 domain of hnRNP A1 was used. One dimensional 1H NMR and differential scanning calorimetry was used to define the structural and thermodynamic stability of each mutant, while electrophoretic mobility shift assays and isothermal titration calorimetry provided additional insight into the binding mechanism.
Keywords: HIV, alternative splicing
Abstract:
Human lysyl aminoacyl tRNA synthetase (LysRS) is an essential protein biosynthesis enzyme that has a wide range of other, auxiliary functions ranging from pro-inflammatory response to chaperoning of the HIV-1 reverse transcription primer molecule, tRNALys,3, during packaging of new HIV-1 particles (1, 2). An important characteristic of higher eukaryotic forms of LysRS is the appendage of an N-terminal domain to the highly conserved anticodon binding (ACB) and catalytic domains. The function of this N-terminal domain has been proposed to enhance the overall RNA binding affinity which may contribute to the HIV-1 reverse transcription primer uptake and possibly modulate LysRS binding to other proteins (3, 4, 5); however, its structure still remains unknown. Here, we present preliminary results of our NMR study to better elucidate the role and potential structure of this appendage domain. Based upon the assigned frequencies of the NMR resonances of this protein, the isolated N-terminal domain is mostly unstructured in solution, in contrast to its secondary structure that is predicted based upon its amino acid sequence. However, this domain does adopt a 27 residue long helical structure but only upon binding to a RNA ligand corresponding to the anticodon stem-loop of the cognate human tRNALys,3. Furthermore, fluorescence anisotropy and EMSA binding studies show that the N-terminal domain of human LysRS does indeed enhance the overall RNA binding affinity of the ACB domain. NMR titration studies using different forms of the N-terminal domain protein via segmental labeling (6) reveal that RNA binding by this N-terminal domain is cooperative with the adjacent ACB domain. Potential RNA binding site of the N-terminal domain is also discussed.
References:
Reference:
1) Park SG, Kim HJ, Min YH, Choi E-C, Shin YK, Park B-J, Lee SW and Kim S (2005) PNAS 102(18):6356-6361
2) Cen S, Khorchid A, Javanbakht H, Gabor J, Stello T, Shiba K, Musier-Forsyth K and Kleiman L (2001) Journal of Virology 75:5043-5048
3) Francin M, Kaminska M, Kerjan P and Mirande M (2002) The Journal of Biological Chemistry 277:1762-1769
4) Cen S, Javanbakht H, Niu M and Kleiman L (2004) Journal of Virology 78:1595-1601
5) Guzzo C, Yang D (2008) Biochemical and Biophysical Research Communications 365:718–723
6) Refaei M, Combs A, Kojetin D, Cavanagh J, Caperelli C, Rance M, Sapitro J and Tsang P (2011) Journal of Biomolecular NMR 49:3-7
Keywords: LysRS, HIV-1, RNA-protein complex
Abstract not available online - please check the printed booklet.
Abstract:
The bacterial ribosome is the target for a number of antibacterial drugs; however, drug resistance in bacteria has increased significantly in recent years. Therefore, novel methods for overcoming the resistance problem are needed. Towards understanding more clearly the function of individual ribosome components in protein synthesis, in vivo genetic studies on a region called helix 69 (H69) were carried out.1 Mutants A1912G, A1917G, and A1919G strongly inhibited bacterial growth and caused inefficient in vitro translation. Residues A1912 and A1917 of H69 were shown to be essential for ribosome function. In this study, in vitro biophysical studies on H69 were carried out in which the relevant nucleotides from the genetic studies were replaced. The goal was to investigate the effects of functionally deleterious mutants on H69 structure, and to understand how these particular nucleotides play roles to either stabilizing or destabilizing H69. The stabilizing effects as well as pH dependence of the structures of mutant H69 RNAs (A1912G, A1918G, and Ψ1917C) were compared to wild-type H69 (a construct that contains three pseudouridine modifications at positions 1911, 1915, and 1917) by using thermal melting analysis and circular dichroism spectroscopy. The results to be presented correlate with the in vivo genetic studies. Our long-term goal is to use these results in combination with drug-binding studies to better understand drug resistance, and to develop improved compounds that take advantage of the genetic information.
References:
1. Liiv, A.; Karitkina, D.; Maivali, U.; Remme, J., Analysis of the function of E. coli 23S rRNA helix-loop 69 by mutagenesis. BMC Mol. Biol. 2005, 6, 18.
2. Hirabayashi, N.; Sato, N. S.; Suzuki, T., Conserved loop sequence of helix 69 in Escherichia coli 23 S rRNA is involved in A-site tRNA binding and translational fidelity. The J. Biol. Chem. 2006, 281, 17203.
Keywords: Ribosome, Helix 69
Abstract:
During translation, the ribosome contributes to the accuracy of aminoacyl(aa)-tRNA selection during two phases of decoding. During the first phase, initial selection, aa-tRNAs are delivered to the ribosome by elongation factor (EF) Tu. EF-Tu must hydrolyze GTP in order for subsequent steps of decoding to proceed, so the ribosome stimulates GTP hydrolysis for EF-Tu complexes carrying a cognate aa-tRNA, allowing those tRNAs to be preferentially incorporated. In the second decoding phase, proofreading, cognate aa-tRNAs are released from EF-Tu and rapidly accommodated into the 50S A site to undergo peptidyl transfer whereas near-cognate aa-tRNAs are almost always rejected.
Recently, we have identified a number of mutations in the 16S rRNA that significantly reduce the accuracy of aa-tRNA selection. Most of the mutations are located at the interface of the ribosomal shoulder domain with other regions of the ribosme. These mutations stimulate GTP hydrolysis by cognate and near-cognate aa-tRNA, reducing the accuracy of initial selection. Additionally, our data show that many of these mutations significantly affect the accuracy of the proofreading phase. We will present evidence of how these mutations influence proofreading by affecting rates of individual steps of this mechanism. These data implicate specific areas of the ribosome in decoding and provide insight into the molecular mechanisms of antibiotics that inhibit the accuracy of translation.
Keywords: Translation, Ribosome, Decoding
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 tRNAHis guanylyltransferase (Thg1) superfamily was first discovered in yeast to catalyze the post-transcriptional addition of an essential tRNAHis identity element required for HisRS recognition, a single G-1 nucleotide found at the 5’ end of almost all tRNAHis species. In most Eukarya, including yeast, this reaction forms a non-templated G-1 -A73 base pair. Previously, an alternative activity of yeast Thg1 (yThg1) was demonstrated with A73C or A73G tRNAHis substrates, where the 3’ N73CCA end was used as a template for a 3’-5’ polymerization reaction. It was predicted that this reverse polymerization was remnant of an ancestral activity, and here we show a similar occurrence in DdiTLP4, a homologue of yThg1 found in the slime mold Dictyostelium discoideum. Unlike yThg1, DdiTLP4 is predicted to participate in cytoplasmic repair of tRNA 5’ ends and has robust in vitro tRNA repair capabilities. Under limited ATP concentrations, however, DdiTLP4 catalyzes templated polymerization much like the activity described for yThg1. More importantly, we observe excessive polymerization reactions on 5’ truncated tRNA’s, the predicted substrate for DdiTLP4, as well as full length tRNAs. This suggests that the polymerization reaction may occur in vivo, yet the resulting tRNAs would contrast with the current understanding of tRNA secondary structure and function. However, we identify a mechanism where ATP is used both as a substrate and an effector molecule to control DdiTLP4 activity and inhibiting excessive nucleotide addition to full-length tRNA substrates. These result point to a novel mechanism developed by DdiTLP4 to sequester an unwanted and possibly ancestral activity.
References:
Jackman JE, Phizicky EM 2006b. tRNAHis guanylyltransferase catalyzes a 3′–5′ polymerization reaction that is distinct from G−1 addition. Proc Natl Acad Sci 103: 8640–8645.
Keywords: tRNA Biology, Thg1
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
Small interfering RNAs (siRNAs) are double stranded RNAs that can target complementary mRNAs for degradation through the RNA interference (RNAi) pathway. Recently it was shown that circularization of the free ends of the siRNAs increase the stability and therapeutic window of RNAi knockdown, though improvements in approaches and yields of circularization are still desirable. We show that the copper catalyzed azide-alkyne cycloaddition (CuAAC) reaction is an efficient and straightforward method of creating synthetic dumbbell shaped siRNAs with click-circularized ends, for effective and stable knockdown in S2 cells. We are also exploring obtaining the dumbbell shaped circularized RNAs through transcription, using a 5'-deoxy-5'hydrazinylguanosine (hydrazine) initiator to functionalize the 5'-terminus. A periodate reaction to render the 3'-end reactive to the 5'-terminus will close the loop and circularize the RNA. These methods provide alternate and efficient approaches to circularized siRNAs.
Keywords: siRNA, Click ligation
Abstract:
Thermus thermophilus is a gram-negative thermophilic bacteria that has served as the model organism for recent studies into ribosome structure and function. Unlike a majority of bacteria, Thermus does not posses the hypermodified nucleoside queuosine (Q) within its tRNAs. In other bacteria, tRNAs coding for asparagine, aspartic acid, histidine and tyrosine contain Q, modified in place of guanosine, at position 34 – the wobble position. While the exact function of Q is still not understood, it appears that this modification may enhance translational fidelity through reading frame maintenance or improved decoding. Before undertaking a detailed study into the significance of queuosine’s absence in Thermus, we first sought to determine whether some other modified nucleoside is found at the wobble position of these four tRNAs. To answer this question, individual tRNAs from Thermus were purified using hybridization probes attached to biotinylated streptavidin beads. These purified tRNAs were digested with RNase A or T1 and the resulting oligonucleotides were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Mass spectrometry sequencing of the anticodon region of these four tRNAs reveals each to contain the genome-encoded guanosine, with no evidence that other modifications are found in place of queuosine for Thermus tRNAs. In addition to presenting the data characterizing the anticodon sequences for these four tRNAs, additional sequence information from each will be presented, including placement of modified nucleosides found at other sequence locations.
Keywords: Thermus Thermophilus, tRNA, queuosine
Abstract:
Pre-mRNAs undergo splicing to remove introns, non-protein coding regions, and religate exons, protein coding regions. This process is catalyzed by the spliceosome, which consists of five small nuclear ribonucleoprotein particles (snRNPs: U1, U2, U4, U5 and U6) and numerous protein factors. Proper assembly of spliceosomal components is critical for function, and thus, defects in assembly can be lethal. Several spliceosomal proteins facilitate structural rearrangements important for spliceosomal assembly and function. Prp24 is an essential factor in U6 snRNP assembly, and it has been proposed to assist in U4/U6 formation and unwinding. Here, we address the question whether Prp24 affects the U2/U6 complex dynamics. Using single-molecule Fluorescence Resonance Energy Transfer (smFRET), we have previously shown that a minimal U2/U6 complex from yeast can adopt at least three distinct conformations in dynamic equilibrium. Our new single molecule data show that Prp24 unwinds U2 from U2/U6 complex and stabilizes U6 in a low FRET conformation. We also show that the RNA Recognition Motifs of Prp24 affect the binding affinity of Prp24 for U6 and unwinding activity. We propose that Prp24 plays an important role in U2 and U6 snRNP recycling by dissociating the U2/U6 complex.
Keywords: Prp24, U2U6, smFRET
Abstract:
All biological processes take place in highly crowded cellular environments. However, how molecular crowding agents affect the folding and catalytic properties of RNA molecules remains largely unknown. Here, we have combined single-molecule fluorescence resonance energy transfer (smFRET) and bulk cleavage assays to determine the effect of a molecular crowding agent (polyethylene glycol, PEG) in the folding and catalysis of a model RNA enzyme, the hairpin ribozyme. Our single-molecule data reveal that PEG favors the formation of the docked (active) structure by increasing the docking rate constant with increasing PEG concentrations. Furthermore, Mg2+ ion induced folding in the presence of PEG takes place at ~10-fold lower concentrations, near the physiological range (~1 mM). Lastly, bulk cleavage assays show that the ribozyme’s activity accelerates by ~10-fold in the presence of the crowding agent. Our data show that molecular crowding agents can affect the structure, stability and function of the RNA enzymes, such as the hairpin ribozyme. We propose that crowding agents play an important role in stabilizing and accelerating the active structure of RNA enzymes in vivo.
Keywords: Ribozyme, smFRET, Docking and Catalysis
Abstract:
The spliceosome is a large, RNA-protein complex that catalyzes pre-mRNA splicing during mRNA maturation. The RNA components (small nuclear RNA; snRNAs) of the spliceosome have been well studied and are believed to be involved in the splicing catalysis. Although proteins are essential for splicing, they may not be directly involved in catalysis. Among hundreds of proteins, Prp8 is the only protein that interacts with all the catalytically important snRNAs. Therefore it is hypothesized that Prp8 may catalyze splicing either by directly participating in catalysis or by stabilizing the conformation of the catalytically active spliceosome. In order to test whether or not Prp8 stabilizes the active site conformation, we carried out single-molecule fluorescence resonance energy transfer (smFRET) experiments with catalytically important snRNAs U2 and U6 and Prp8. We observed that, in the presence of Prp8, the population of the high FRET conformation of U2-U6 that is thought to be the active conformation increased, indicating that one of the functions of Prp8 would be to stabilize the active site conformation of the spliceosome.
Keywords: Prp8, smFRET, spliceosome
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
During ribosome biogenesis, structural rearrangements in the pre-rRNA and in trans acting snoRNAs are thought to help the small subunit (SSU) processome coordinate the sequential cleavage events that liberate the SSU precursor. Cleavage or protein binding may trigger structural changes that transition the RNA from one state to the next, thereby permitting the next step in processing or assembly. Here we focus on how a U3 specific protein Imp3p mediates one of the three sites of hybridization between the pre-rRNA and U3 snoRNA, which is required for the pre-rRNA cleavages (A1 and A2) that release the 20S small subunit rRNA precursor. In contrast to two of the U3-pre-rRNA duplexes, the third, designated the U3-18S duplex, is only observed in the presence of protein using minimal substrates in vitro. Duplex formation is blocked because prior to hybridization bases from both duplex strands are buried: one in an A/A’ stem-loop structure in U3 snoRNA and the other in helix 1 (H1) of the 18S region of the pre-rRNA. To test whether protein binding removes hybridization barriers by unfolding relevant stem-loop structures in the U3 and pre-rRNA we assessed base accessibility by chemical probing with CMCT in the presence and absence of protein. We show that Imp3p alone is sufficient to unfold the stable U3 structure and to mediate rapid U3-18S hybridization. Moreover, we show that Imp3p binds to a 257 nt pre-rRNA fragment containing H1. Addition of Imp3p results in a marked increase in accessibility of the bases in H1 involved in U3-18S hybridization and bases that flank the A1 cleavage site. The increased base accessibility of H1 provides evidence that Imp3p binding unfolds this stem structure, presumably to expose bases for U3-18S duplex formation, just as Imp3p binding unfolds the U3 stem structure. Perhaps the late arrival of Imp3p during assembly of the SSU processome mediates U3-pre-rRNA hybridization to signal subsequent A1 (and possibly A2) cleavage.
Keywords: Chaperone, Conformational Change, Ribosome biogenesis
Abstract:
Over the past decade, single-molecule studies with extrinsic fluorescent dyes have provided exciting new insight into nucleic acids folding and accompanying conformational changes. However, the use of ‘semi-intrinsic’ fluorescent nucleotide analogues, such as 2-aminopurine (2AP) and Pyrrolo-C (PC), has not been reported at the single-molecule level despite their high photon rate. For instance, 2-aminopurine has a photon rate of 2 kHz. Numerous ensemble-averaged studies have proven the utility of these probes to decipher mechanistic steps that involve local rearrangements and base flipping.
Here, we present a new single-molecule assay that takes advantage of the fluorescent properties of 2AP and PC to study local base dynamics. We utilize a novel ‘Click’ chemistry based single-molecule immobilization methodology that enables sufficient minimization of background fluorescence to allow single-molecule detection. We have incorporated 2AP into single- and double-stranded DNA molecules and reveal, in real time, local base dynamics. We are also using PC to study similar motion in RNA molecules. Our results show that 2AP and PC fluoresce steadily, and without blinking, when not stacking with neighboring bases. Removing molecular oxygen by degassing the buffers results in long-lived fluorescence emission lasting for minutes. We anticipate that this strategy will provide new insights into the local dynamics of nucleic acids and nucleic acids-protein complexes.
Keywords: 2-aminopurine, Pyrrolo-C, Base dynamics
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
Systemic trafficking of RNA is a key process for RNA-based pathogens to establish widespread infection throughout a host organism; however, its mechanism remains poorly understood. The Potato Spindle Tuber Viroid (PSTVd) is a plant pathogen composed solely of single-stranded, noncoding RNA. PSTVd contains a number of loops and bulges that form highly-structured three-dimensional motifs essential to its ability to traffic. It is hypothesized that these unique RNA motifs are responsible for mediating trafficking across cellular boundaries, but the mechanism responsible remains unknown. Analysis of a trafficking-defective mutant of PSTVd Loop 19 led to the detection of rapid in vivo evolution by which the mutant regained capability of systemic trafficking. This gain-of-function mutation, termed Loop 19*, acquired a de novo secondary structure similar to Loop 19, in a one base-pair shifted genomic location. Determining the structures of these loops would allow for the identification of the non-canonical interactions present within the loops. To reduce system complexity, we have designed and produced short oligonucleotides that resemble the loops and are amenable to analysis by Nuclear Magnetic Resonance (NMR). Initial 1H NMR studies confirmed correct foldedness of each loop and reinforced the potential for further NMR studies. 1H-1H NOESY experiments allowed for imino proton assignments. Currently, 13C/15N-labeled samples are being produced by in vitro transcription for multidimensional heteronuclear NMR experiments and structure calculation. Using PSTVd as a model to study these RNA motifs has broad implications for studying the structure-function relationships of RNA motifs in other biological processes.
Keywords: RNA, NMR, viroid
Abstract:
The recent transcriptome analyses have revealed that a large numbers of long non-coding RNAs (lncRNAs) are transcribed from the higher eukaryotic genomes, and that many of them originate within the intergenic region of protein-coding genes. We have identified BORG lncRNA, which plays a crucial role in neuronal differentiation and reprogramming. The mouse BORG RNA is an intergenic transcript, resulting from splicing of two introns from three exons. Analysis of the BORG gene in mouse indicates the presence of several retrotransposon elements within and near the BORG locus. Interestingly, the transcriptional start site of the BORG gene is located within a repeat element. Further, all four exon-intron junctions in the BORG gene fall within repeat elements. Finally, the polyadenylation signal of BORG is also derived from a retrotransposon repeat element. The mature, spliced BORG RNA contains five repeat elements of LTR and SINE subtypes with 40% of nucleotides within BORG derived from repeat elements. Thus, it seems that the evolution of BORG RNA is largely the result of transposition events. Interestingly, these repeat elements seem to be conserved among mammals. However, the role of repeat elements in evolution of long non-coding RNAs in different species remains poorly understood. In this study, we have analyzed the relationship between gene architecture and repeat elements using a database of known mRNA and lncRNAs in human and mouse. The results of this analysis in BORG RNA and other protein-coding and non-coding RNAs will be discussed.
Keywords: Long non-coding RNA
Abstract:
In mammals, pre-rRNA processing happens almost exclusively post-transcriptionally. This is not the case in rapidly dividing yeast, as the majority of pre-rRNA is processed co-transcriptionally. The process of ribosome assembly is thought to be driven, in part, by the hierarchical association of assembly factors and r-proteins. It is well established that there are groups of proteins that associate with nascent ribosomes early in assembly, during the middle of assembly, or only at the end of assembly. Here, in the course of studying Drs1, we find that disruption of ribosome assembly results in a significant shift from co- to post-transcriptional pre-rRNA processing and breakdown of the traditional assembly hierarchy. Previously Drs1 was shown to be required for production of 60S subunits and function in 27SB pre-rRNA processing. Here we show that depletion of Drs1 affects earlier steps of pre-rRNA processing and that Drs1 functionally and physically interacts with the Nop7-subcomplex. Most important, we show that depletion of Drs1 and subsequent disruption of ribosome biogenesis causes pre-rRNAs to be processed post-transcriptionally. We describe a previously unreported phenotype, in which late-binding 66S assembly factors associate with early pre-ribosomes containing 35S pre-rRNA. The shift from co- to post-transcriptional pre-rRNA processing, and the ability of late-associating proteins to bind pre-ribosomes very early in assembly drastically changes the current views on the hierarchy of ribosome assembly.
Keywords: ribosome assembly, pre-rRNA processing, co-transcriptional processing
