RRM
2010 Rustbelt RNA Meeting
RRM
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Poster abstracts
Abstract not available online - please check the printed booklet.
Abstract:
Polyadenylation is one of the most important post-transcriptional processes that result in the formation of mature mRNA. Identification of specific regions on RNA, binding to the RNA and its interaction with protein complexes that associate with other protein complexes play key role in 3’-end processing. In plants, this process is guided by Cis-regulatory elements like far-upstream element (FUE), near-upstream elements (NUE) and cleavage/ polyadenylation site. The multi-subunit protein complexes like Cleavage and polyadenylation specificity factor (CPSF), Cleavage stimulation factor (CstF), Cleavage factors and Poly(A) Polymerase are important factors for polyadenylation in eukaryotes. In Mammals, the Cleavage stimulation factor (CstF) is made up of three subunits CstF 77, CstF64 and CstF 50.
Here we focus on Arabidopsis (FY) orthologue of the essential yeast subunit Pfs2p. FY (At5g13480) is a known 3’-end processing factor and plays a key role in the auto regulation of FCA and interacts with FCA for chromatin silencing that targets FLC (flowering locus C), a repressor of flowering. We established a tethering assay to study the role of FY as a general 3’-end processing factor using transients assays by Agro-infiltrations. Initial results using this assay suggest that tethering of FY to an RNA does not promote polyadenylation of the RNA. Studies are under way to confirm these results, using 3’-RACE and tethering assays involving other polyadenylation factor subunits.
FY also interacts with other polyadenylation factors like CstF77, CPSF 100, CPSF160, CPSF 30 and Fip1 in carrying out the polyadenylation reaction. Our hypothesis is that if FY is unable to bring 3’-end processing complex to the RNA via tethering, it might be one of these other proteins that accomplish this task.
References:
1. Li, Q. and A. G. Hunt (1997). The Polyadenylation of RNA in Plants. 115: 321-325.
2.
Y Takagaki, J L Manley, C C MacDonald, J Wilusz, and T Shenk (1990) A multisubunit factor, CstF, is required for polyadenylation of mammalian pre-mRNAs.
3. Richard M. Amasino (2003). Flowering time: a pathway that begins at the 3 end. vol 13 Issue 17.
4. David Manzano, Sebastian Marquardt, Alexandra M. E. Jones, Isabel Bäurle, Fuquan Liu, and Caroline Dean-Altered interactions within FY/AtCPSF complexes required for Arabidopsis FCA-mediated chromatin silencing.PNAS 2009 106 (21) 8772-8777;
5. Arthur G Hunt*1, Ruqiang Xu2,3, Balasubrahmanyam Addepalli1,Suryadevara Rao1, Kevin P Forbes1,4, Lisa R Meeks1,5, Denghui Xing2,Min Mo2,6, Hongwei Zhao2, Amrita Bandyopadhyay1,Lavanya Dampanaboina1, Amanda Marion1, Carol Von Lanken1 and Qingshun Quinn Li*2.BMC Genomics 2008, 9:220 doi:10.1186/1471-2164-9-220
Keywords: Tethering, RACE (Rapid Amplification of cDNA Ends), Polyadenylation
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
CPSF30 is a component in 3’ RNA processing that has been well characterized in the model species Arabidopsis thaliana (1). In this study the gene for CPSF30 has been cloned out of Medicago truncatula and Medicago sativa. A large region within the gene exhibits significant conservation between A. thaliana and both Medicago species. This area of the gene was cloned out and 5’ and 3’ RACE were used to get each full length gene. The Medicago genes were cloned into a protein expression vector. The vector was used to translate each gene into its corresponding protein and each protein was purified. CPSF30 from A. thaliana has been shown to possess three CCCH type zinc fingers, with the first implicated in RNA binding and the third in RNA cleavage (1). Amino acid alignments showed the zinc finger regions were conserved and it was hypothesized that CPSF30 from both Medicago species would bind RNA and cleave RNA. Electromobility shift assays were used to determine that RNA binding did occur. RNA nuclease activity in Medicago is still being tested. CPSF30 knockout studies in A. thaliana have yielded some consistent phenotypes that are still being characterized and explored. One such characteristic is an elevated resistance to oxidative stress (2). CPSF30 knockouts have been produced in Medicago sativa. Preliminary data suggests that the absence of CPSF30 expression results in a distinct root phenotype. The CPSF30 knockout plants were crossed with a cultivar, Spreader4, which is an agriculturally relevant variety. It has been determined that the knockout vector is present in the hybrid progeny and phenotypes are still being evaluated.
References:
(1) Delaney KJ, Xu R, Zhang J, Li QQ, Yun KY, et al. (2006) Calmodulin interacts with and regulates the RNA-binding activity of an Arabidopsis polyadenylation factor subunit. Plant Physiol 140: 1507–1521.
(2) Zhang J, Addepalli B, Yun K-Y, Hunt AG, Xu R, et al. (2008) A Polyadenylation Factor Subunit Implicated in Regulating Oxidative Signaling in Arabidopsis thaliana. PLoS ONE 3(6): e2410. doi:10.1371/journal.pone.0002410
Keywords: CPSF30, Medicago
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:
In splicing, introns are removed and exons are joined together to generate the correct RNA for translation. The byproduct of splicing is the removed lariat intron. In eukaryotes, lariat introns are branched structures with highly conserved nucleotide sequences around an adenosine branch-point residue that has a 2',5'-phosphodiester linked branch. Branched RNA is a mimic of the natural lariat RNA and known synthetic and DNAzyme based methods to generate branched RNA either do not provide access to natural consensus branched RNA sequences or are limited in the ability to site specifically modify the RNA sequences. We report a simple scheme for the solid-phase synthesis of branched RNA that is not limited to specific sequences and is readily amenable to modified residues and linkages. Using this scheme, the natural yeast branched RNA consensus sequence was synthesized and characterized. This branched RNA is efficiently debranched by yeast debranching enzyme Dbr1. Additionally, unnatural branches including 2',3'-phosphodiester linkages as well as DNA-RNA chimeric analogues were synthesized. The ability to generate branched RNA consensus sequences and to site specifically modify the branched RNA are tools that will help in the study of debranching enzyme and splicing.
Keywords: solid-phase synthesis, branched RNA, lariat intron
Abstract:
RNA editing describes the process in which individual or short stretches of nucleotides in a messenger or functional RNA are inserted, deleted, or substituted. Here we study the mitochondrial genomes of two related organisms with insertional RNA editing, Physarum polycephalum and Didymium iridis. The combination of the two genomes can provide conservation information which is not contained in either of the genomes alone. Sixteen genes and their mRNA sequences from the two organisms are studied: atp1, apt6, atp8, atp9, cox1, cox2, cytb, nad4L, nad6, nad7, rpL2, rpL16, rpS3, rpS7, rpS12, and rpS19. We identify the “C” insertional RNA editing sites shared by the two organisms. Then we calculate the probability for observing increased sequence conservation in the vicinity of the shared insertional editing sites based on the background frequencies. We do not find any statistically significant variations, which implies that the information on editing site is not contained within the sequence in the immediate vicinity of the editing site. In addition, we examine the codon position bias in insertional RNA editing of these two organisms. We find a strong relationship between the strength of the codon bias and the overall sequence conservation in the two organisms: more conserved genes tend to have more significant codon bias. This reinforces the theory that codon bias is a consequence of evolutionary pressure on the protein sequence.
Keywords: RNA editing, sequence conservation, codon bias
Abstract not available online - please check the printed booklet.
Abstract:
The Capsid (CA) domain of HIV-1 Gag polyprotein plays a critical role in both the early and late phase of viral replication. In the early phase of HIV-1 infection the CA coat disassembles and releases the RNA genome into the host cell where it is reverse transcribed and integrated into the host genome. Upon translation, the CA domain of the Gag polyprotein binds to human Lysyl-tRNA synthetase (LysRS) to form a tRNALys primer packaging complex. Further, as the virus buds from the host cell membrane during the late phase, the proteolytic cleavage of the Gag protein leads to the formation of the characteristic conical core formed by closed hexameric array of the viral CA protein in the mature virus. Being versatile in nature makes CA an important antiviral target for disrupting the assembly of HIV-1. Small-molecule inhibitors against HIV-1 CA, commonly referred to as capsid assembly inhibitors (CAI), have potential clinical applications and hence may serve as useful molecular probes in biomedical research. In this work, a robust method has been employed for the high-throughput synthesis, screening and identification of cyclic peptidyl ligands against macromolecular targets. Support-bound cyclic peptide libraries containing randomized amino acid sequences and different ring sizes (theoretical diversity of 1.2 × 107) were synthesized and screened against CA and the monomeric form of the CA-terminal domain (WM-CA CTD). Cyclic Peptide 2 and 4 (CP2, CP4) out of six selected cyclic peptides showed strong binding (KD ~ 500 nM) to both CA and WM-CA CTD in vitro. The scrambled variants of CP 2 and CP 4 resulted in no binding to either CA or WM CA-CTD suggesting a sequence specific mode of interaction. CP2 and CP4 also inhibited LysRS- CA or WM CA-CTD interaction with an IC50 value of 1 µM. Furthermore, preliminary chemical-shift perturbation NMR analyses suggest that CP 4 binds to a site proximal to helix 4 of the CA-CTD, which is the known site of LysRS interaction.
Keywords: Lysyl tRNA Synthetase, HIV-1, Capsid
Abstract:
The Neurofibromatosis Type I (NF1) pre-mRNA undergoes alternative splicing. NF1 exon 23a is an alternative exon that shows differential expression in brain versus other tissue types. Exon 23a, which is skipped in neuronal tissues and included in other tissue types falls within the best-characterized domain of the protein, the GAP-related domain. Through its GAP-related domain, NF1 negatively regulates Ras signaling, thus controlling cell growth and proliferation. The type II isoform, which contains exon 23a, is ten times weaker in regulating Ras than the type I isoform, in which exon 23a is skipped. Mice in which the balance of the two NF1 isoforms has been disrupted show learning disabilities.
In our lab, we have identified several factors that regulate the NF1 exon 23a inclusion. We have shown that the Hu proteins and the CUG-BP and ETR-3 like factors (CELF proteins) promote NF1 exon 23a skipping, while TIA-1 and TIAR promote exon 23a inclusion (ref. 1 & 2). The Muscleblind-like (MBNL) proteins are additional candidates for NF1 splicing regulation, and we hypothesize that they promote NF1 exon 23a inclusion. MBNL proteins are known to act as antagonists to the CELF proteins in at least six pre-mRNA targets, although the two families also regulate distinct targets. MBNL proteins bind to the YGCU(U/G)Y motif (Y, pyrimidine) with a preference for stem-loop RNA secondary structures.
There are two potential MBNL binding motifs in the intronic sequence upstream to NF1 exon 23a, and this RNA sequence is predicted to form a stem-loop structure. Point mutations that disrupt the potential MBNL binding motifs in NF1 led to decreased inclusion of exon 23a from an NF1 splicing reporter. Over-expression of MBNL1 promoted NF1 exon 23a inclusion, in a low MBNL expressing cell line, suggesting that they are positive regulators. Interestingly, over-expression of MBNL proteins partially rescues the increased skipping of NF1 exon 23a that results from the over-expression of CELF proteins in HeLa cells. Binding of recombinant MBNL1 protein to the NF1 pre-mRNA was confirmed by UV crosslinking. Future work will focus on the regulation of this event by MBNL and how MBNL and CELF proteins function as antagonists in this system. In addition the secondary structure of this region will be probed using in vitro RNA structure probing methods.
References:
1. Zhu, Hui et al., 2008. Regulation of Neuron Specific Alternative Splicing of Neurofibromatosis Type I Pre-mRNA. Mol Cell Biol. 28: 1240-1251.
2. Barron, Victoria A. et al., 2010. The Neurofibromatosis Type I Pre-mRNA is a Novel Target of CELF Protein-Mediated Splicing Regulation. 38: 253-264.
Keywords: Muscleblind Proteins, Alternative Splicing, Neurofibromatosis Type I
Abstract:
Of most metazoan genes, one of the most essential and critical step in gene expression is the removal of intervening sequences or introns by splicing from the primary transcripts. Intron removal is accomplished by a distinct set of spliceosome consisting of major and minor class of spliceosomal small nuclear RNAs (snRNAs) whose intermolecular and intramolecular sequential RNA-RNA base pairing interactions are often analogous. In the minor or U12-dependent spliceosome U11, U12, U6atac and U4atac are unique snRNAs and are essential for the splicing of U12-dependent intron. We are studying sequential base-pairing interactions between U4atac:U6atac and U6atac:U12 snRNAs to understand the role and requirement of a conserved intermolecular helix I. Single and double nucleotide mutations revealed base-pairs which are essential and required for the formation of helix I. Also, for the first time our data produces the evidence of in vivo intermolecular helix I of U6atac:U12 snRNAs.
Keywords: U12- dependent pre-mRNA splicing, intermolecular helix I, Single and double nucleotide mutations
Abstract:
A large number of microRNAs (miRNAs) are encoded in introns of protein coding genes. The processing of these miRNAs takes place from the primary and precursor stem Loop expressed cotranscriptional with the host precursor mRNA. Experimental evidence, thus far suggest that miRNA processing is independent of the host pre-mRNA processing. Previous these studies overlooked the pre-mRNA processing and its subtle effect on mature miRNA production and target mRNA expression. In this study, we investigated if mutations in the 5’ splice site of a miRNA intron influence the production of the mature resident miRNA and alter its target gene expression. We generated a series of point mutations starting from the position 1 to 7 of MYH6 intron 29 which host miR208a. Our preliminary data indicated that 5’ splice site mutations affects the mature miRNA production and its target mRNA modulation in vivo.
Keywords: intronic microRNAs, MYH-6, splicing
Abstract:
X chromosome inactivation is a dosage compensation strategy that mammals use to balance the protein levels expressed from X chromosome-linked genes between females (XX) and males (XY). This process strategically inactivates one of the X chromosomes in female mammals. Most genes from this inactivated chromosome are silenced except X inactivation specific transcript (Xist) gene. At the onset of X inactivation, Xist gene is transcribed into a large non-coding RNA, which wraps the X-chromosome that is going to be inactivated in cis and recruits certain protein complexes to assist in the inactivation event. The identities of these interacting proteins have not been well defined mainly because of the difficulty to isolate an ¡°active¡± Xist-RNP particle. In order to identify these RNA interacting proteins in vivo, a 14-mer peptide nucleic acid (PNA) complementary to a unique sequence located in the conserved region of Xist RNA is synthesized and covalently linked to a nuclear localization signal (NLS) peptide. The PNA-NLS is incubated with a female human breast cancer cell line and a PNA-assisted identification of RNA binding proteins (PAIR) technique is used to search for X inactivation related proteins. Identification of Xist RNA binding proteins will be used to elucidate the mechanism of X-inactivation.
References:
Current Opinion in Genetics & Development, 2009, 19:122¨C126
Nature protocols, 2006, 1(2), 920-927
Keywords: Xist, PNA, PAIR
Abstract:
Polypyrimidine Tract Binding protein (PTB) is a key alternative splicing factor involved in exon repression and Fox-1 (feminizing on X) is a splicing enhancer. It has been proposed that PTB acts by looping out exons flanked by pyrimidine tracts. Fox-1 has been proposed to bind a UGCAUG element to activate exon inclusion, but the mechanism of Fox binding to RNA and its implications for alternative splicing regulation are still poorly understood. Here, we present fluorescence, NMR and in vivo splicing data that directly support a looping mechanism for PTB. We show that PTB loops the RNA by binding two distant pyrimidine tracts with RNA binding domains (RBD) 3 and 4 of PTB to bring the 5’ and 3’ ends in close proximity. Looping efficiency depends on the length of the intervening sequence with preference for a spacer at least 15 nucleotides between the pyrimidine tracts. Furthermore, RBDs 3 and 4 work synergistically for efficient RNA looping in vivo. Finally, our preliminary data suggest that Fox-1 acts as a splicing enhancer by displacing one of PTB’s RBDs from the RNA to abrogate looping. This work makes significant progress in understanding the mechanisms of alternative splicing regulation by PTB and Fox-1.
Keywords: Alternative Splicing, FRET, PTB
Abstract:
Splicing is an essential step in the maturation reaction of eukaryotic pre-mRNA, in which intervening sequences (introns) are removed from the coding sequences (exons). The spliceosome is a dynamic assembly of five small nuclear RNAs and a large number of proteins that catalyzes splicing. U2 and U6 are the only two spliceosomal snRNAs strictly required for both steps of splicing, and they form an RNA complex using base pairing. Major conformational changes are expected to take place during the assembly and catalysis of the spliceosome.
We have developed a Single-Molecule Fluorescence Resonance Energy Transfer (smFRET) assay to study the structural dynamics of a protein-free U2/U6 complex from S. cerevisiae. Our previous data have revealed a two-step large amplitude conformational change of the U2-U6 complex. The first step is a Mg2+-induced conformational change where helix III and the U6-ISL are in close proximity in low [Mg2+] and separated in high [Mg2+]. The second rearrangement corresponds to the formation of the highly conserved Helix IB.
According to the recent crystal structure of the group II intron and given the similarity between the group II intron and the spliceosome, we hypothesize that similar base triple interaction may exist in the U2/U6 complex. Here, we present new experiments that examine the role of the highly conserved base U80 and the ACAGAGA in formation of base triple with the AGC triad. The data show that both U80 and the ACAGAGA loop play an important role in stabilizing the interactions maintaining the high FRET structure. Mutations that disrupt these interactions prevent helix III and the U6-ISL from being in proximity of each other. We hypothesize that in physiological [Mg2+], U80, G52and A53 form base triples with the AGC triad to pull helix III and the U6-ISL together. Our results raise the interesting possibility that this base triple interaction plays an important role in bringing the 5’ splice site and the branched A into close proximity of U80, which may be critical for catalysis.
Keywords: splicing, U2U6, base triples
Abstract:
Micro (mi) RNAs are short ribonucleic acid segments 19 to 24 nucleotides in length. They are non-protein coding RNAs, first discovered in the nematode C. Elegans, and are highly conserved in eukaryotic organisms. The human genome encodes hundreds of miRNAs that target approximately 60% of human genes and are prevalent in many human cell types. MiRNAs post-transcriptionally regulate gene expression by binding to the 3’ untranslated region (UTR) of mRNA. This binding typically negatively affects the expression of the target gene. Our lab has shown that a miRNA, hsa-miR-488* down regulates luciferase activity for plasmids containing androgen receptor (AR) 3’ UTR segment. Using bioinformatics and rational approaches we have identified 43 more potential target genes of miRNA hsa-miR-488*. In this study, we have generated a library of luciferase reporters expressing 3’ UTR segments of potential target genes of hsa-miR-488*. Our data show that hsa-miR-488* can suppress luciferase activity for plasmids containing 3’ UTR target sites of many other genes and suggest negative regulation of multiple genes in prostate cancer. Further studies would determine the role of hsa-miR-488* and its potential array of target genes in prostate cancer development and progression.
Keywords: MicroRNA, Prostate Cancer, Gene Regulation
Abstract:
In vivo, many RNA molecules can adopt multiple conformations depending on their biological context such as the HIV Dimerization Initiation Sequence (DIS) or the DsrA RNA in bacteria. It is quite common that the initial interaction between the two RNAs takes place via complementary unpaired regions, thus forming a so-called kissing complex. However, the exact kinetic mechanism by which the two RNA molecules reach the dimerized state is still not well understood. To investigate the refolding energy surface of RNA molecules, we have developed new technology based on the combination of single molecule spectroscopy with laser induced temperature jump kinetics, called Laser Assisted Single-molecule Refolding (LASR). LASR enables us to induce folding reactions of otherwise kinetically trapped RNAs at the single molecule level, and to characterize their folding landscape. LASR provides an exciting new approach to study molecular memory effects and kinetically trapped RNAs in general. LASR should be readily applicable to study DNA and protein folding as well.
Keywords: single-molecule, temperature jump kinetics, RNA kissing loop interaction
Abstract:
How an RNA itself directly regulates its trafficking between cells remains poorly understood, though the importance of inter-cellular RNA trafficking has been well-established based on the studies on both endogenous and infectious RNAs in plants. Viroids, small but highly structured non-coding RNAs, are excellent models to investigate the molecular mechanism of RNA trafficking. Using Potato spindle tuber viroid (PSTVd) infection of Nicotiana benthamiana as the experimental system, we demonstrated that various RNA loop structures, consisting of non-Watson-Crick base pairs, were essential for PSTVd systemic trafficking. Here, we present our analyses on the role of loop 6, a conserved motif comprising six nucleotides, in regulating inter-cellular RNA trafficking. The tertiary structural model of loop 6 was inferred by comparisons with the X-ray crystal structures of similar motifs in other RNAs. Extensive mutational analyses supported the structural model. Cellular analyses revealed that the maintenance of the tertiary structure of loop 6 is essential for PSTVd to traffic between specific cell types. Our findings support the hypothesis that unique RNA structural motifs mediate trafficking across distinct cellular layers. Furthermore, our approaches should be useful in characterizing the structure-function relationships for other RNA motifs.
Keywords: Viroid, RNA trafficking, RNA structural motif
Abstract not available online - please check the printed booklet.
Abstract:
A strict regimen of highly active antiretroviral therapy (HAART) administered to HIV infected individuals is unable to eliminate latent HIV proviruses that are stably integrated in the genome of a very small population of long-lived memory CD4+ T lymphocytes. When HAART is interrupted, T cell receptor stimulation during foreign antigen presentation induces the reactivation of latent HIV expression. In a latent HIV Jurkat T cell model, initiation of T cell receptor (TCR) signaling stimulates rapid transcription elongation of the HIV provirus. TCR-activated processive proviral transcription is mediated by the MAPK/ERK pathway which induces the release of the HIV Tat elongation co-factor P-TEFb from its inactive nuclear regulatory 7SK snRNP complex. Defining the biochemical mechanisms for the release of P-TEFb from 7SK snRNP and its recruitment to the HIV gene could prove useful in developing therapies that specifically target the reactivation of latent HIV expression.
Keywords: HIV latency; Transcription elongation; P-TEFb, 7SK snRNP; 7SK snRNA
Abstract:
We will display recent results of a physically motivated bioinformatic
search for miRNA as well as deep sequencing data corresponding to small
non-coding RNAs from blood-stage Plasmodium falciparum lysate. While no
small RNA gene regulatory pathway is currently known in Malaria, it is
possible that snoRNA or introns could be processed into functional
regulatory elements akin to microRNA called mirtrons. We searched for
possible microRNA using a novel bidirectional target-finding algorithm
and identified several candidates. Experimental evidence from deep
sequencing indicates that snoRNA are indeed processed into small RNA of
length 16-35nt. We attempt to relate this data to putative gene
regulation pathways in Malaria.
Keywords: Malaria, Deep Sequencing, microRNA
Abstract:
Aminoacyl-tRNA synthetases play an integral part in translation by maintaining fidelity in protein synthesis. They catalyze a two-step reaction wherein an amino acid is first activated in the presence of ATP to form an aminoacyl-adenylate. Then in the second step the activated amino acid is aminoacylated to the end of its cognate tRNA. These enzymes are dynamically active assemblies in which there exists a direct link between protein structure, dynamics, and function. Internal dynamics are crucial for maintaining a protein’s biological function including substrate binding and catalysis. Computational and biochemical strategies have been employed to investigate how a single mutation of a noncatalytic residue can modify internal protein dymanics. Specifically, we examined how the motion of the catalytically significant proline binding loop (PBL) in E. coli prolyl-tRNA synthetase is affected by fluctuations at a distant site. The PBL is a mobile loop that folds over the proline moiety of the activated adenylate upon binding. Normal mode analysis revealed that the G217- and E218-containing segment is engaged in correlated motion with the PBL region (residues 190-210). Essential dynamics studies suggest that mutation of G217 or E218 to alanine has significant effects on the motion of the PBL and the overall dynamics of the protein. Mutations of these two highly conserved residues resulted in altered activity of the protein. Cognate proline activation was decreased by ~45-fold in the E218A mutant and ~7-fold in the G217A mutant. Aminoacylation rates were also reduced up to 3-fold. The combined molecular dynamics and experimental mutational studies suggest that coupled motions facilitate substrate binding and activation by bacterial prolyl-tRNA synthetases.
Keywords: aminoacyl-tRNA synthetase
Abstract not available online - please check the printed booklet.
Abstract:
Neurofibromatosis type 1 (NF1) functions in the regulation of cell proliferation and differentiation, particularly in the nervous system. Mutations in NF1 lead to a dominant disorder with variable phenotypes including benign tumors of the peripheral and optic nerves, café au lait spots, and learning disabilities. NF1 is a tumor suppressor that inactivates the oncogene Ras through its GTPase activating protein (GAP)-related domain (GRD). NF1 alternative exon 23a falls within the GRD, and its inclusion decreases Ras-GAP activity. Exon 23a is mainly included in most tissues but skipped in neurons. Throughout development, exon 23a inclusion is tightly regulated, with a decrease in inclusion that correlates with neuronal differentiation. We hypothesize that regulation of exon 23a inclusion is important for the biological functions of NF1.
Mice lacking exon 23a exhibit learning defects but normal development and tumor incidence. We will further investigate the biological importance of exon 23a inclusion by ablating its regulation. Using a splicing reporter system, we mutated the 5’ splice site and polypyrimidine tract of exon 23a to more closely match consensus sequences. In a variety of cell types, including primary neurons, exon 23a inclusion from the mutant splicing reporter approaches 100%, and is not affected by overexpression of negative splicing regulators from the Hu and CELF families. We targeted these mutations into the endogenous Nf1 locus of mouse embryonic stem (ES) cells, and are currently generating mutant mice. Mutant ES cells show nearly 100% exon 23a inclusion, even after neuronal differentiation. We will examine mutant ES cells for alterations in Ras GAP activity, proliferation, and neuronal differentiation, and mutant mice for phenotypes such as increased tumor incidence and defects in neuronal development. These studies will illustrate the biological significance of this evolutionarily conserved, regulated alternative splicing event.
Keywords: Alternative Splicing
Abstract:
Fidelity during protein synthesis relies on aminoacyl-tRNA synthetases, which are responsible for matching specific amino acids with their cognate tRNAs. Although, synthetases are efficient enzymes, the similar size and volume of many amino acids results in the formation of mismatched amino acid-tRNA pairs. For example, prolyl-tRNA synthetase (ProRS) can mis-activate and mis-acylate tRNAPro with cysteine or alanine. However, bacteria have evolved editing mechanisms to ensure the fidelity of the proline codon. In most bacteria, Ala-tRNAPro is hydrolyzed by a ProRS catalytic domain distinct from the synthetic active site, the insertion domain (INS), via “post-transfer” editing. In contrast, YbaK, a free-standing homolog of the ProRS INS domain, edits Cys-tRNAPro. Interestingly, the complete genomes sequenced to date have revealed that some bacterial ProRSs encode a truncated INS domain that has lost editing capability. However, many of these organisms encode an additional free-standing INS domain homolog. For instance, Caulobacter crescentus (Cc) expresses a truncated ProRS and encodes both YbaK and PrdX. We hypothesize that the latter is a deacylase that hydrolyzes Ala-tRNAPro. We cloned, expressed, purified, and characterized the in vitro activity of Cc ProRS, YbaK, and PrdX and found that Cc ProRS can mis-charge Cys onto tRNAPro, and to a lesser extent mis-acylates Ala. As expected, Cc YbaK showed hydrolytic activity towards Cys-tRNAPro. On the other hand, PrdX can deacylate Ala-tRNAPro and Ala-tRNAAla in vitro. Our data suggest that the fidelity of proline codon translation in Cc relies on the activities of YbaK and PrdX, since Cc ProRS is unable to edit Cys- or Ala-tRNAPro. This study provides the first characterization of an organism possessing a ProRS that lacks post-transfer editing capability, but encodes two free-standing domains that collaborate to maintain fidelity during translation.
Keywords: prolyl-tRNA synthetase, YbaK, PrdX
Abstract:
Androgen receptor (AR), a ligand-dependent transcription factor plays a key role in the development of both androgen-dependent and androgen-independent prostate cancer (PCa). The suppression of AR-mediated signaling is the main therapeutic approach for the treatment of PCa. Currently available AR-targeted therapies cannot completely shut down AR signaling as androgen-independent PCa cells utilize multiple alternative mechanisms to activate AR. Hence, there is a need to develop innovative strategies for targeting AR, which can produce a durable repression of AR activity. Recent studies suggest that microRNAs (miRNAs) can contribute to carcinogenesis by acting as tumor suppressors or oncogenes. Here, we show that miRNAs are involved in the regulation of AR expression and these miRNAs could be exploited for targeting AR in PCa. We have identified a set of “Andro-miRs”, which repress the AR protein expression in PCa cells. One of the miRNAs was found to inhibit the proliferation and enhance apoptosis of PCa cells. Our findings provide insight for utilizing miRNAs as novel therapeutics to target AR in PCa.
Keywords: Prostate cancer, microRNAs, Androgen receptor
Abstract:
Mass spectrometry (MS) gives a unique approach to the problem of RNA sequencing and post transcriptional modification status. In MS analysis individual molecules can be detected and identified by their mass to charge ratio (m/z), thus a RNA can easily be identified based on a theoretical m/z value. The identity of these molecules can further be confirmed and sequenced by the use of MS/MS. Large strands of RNA like tRNA it must be enzymatically digested into smaller fragments prior to analysis. Every enzyme produces different digestion products based on where they cause cleaves. For the detection of more than one tRNA each must have a digestion product with a unique m/z value specific to that tRNA. These digestion products are known as signature digestion products.
High pressure Liquid chromatography (HPLC) is a technique that is often coupled with MS detectors to aid in the analysis of complex samples by separating the components of a mixture. A variation of this technique ultra high pressure Liquid chromatography (UPLC) which utilizes a smaller sorbent size, which has been known to cause better resolution but causes higher back pressure on the instrument. The smaller particles reduce the amount of diffusion during the separation process leading to narrower sample plugs entering the detector.
In this comparison E. coli total tRNA was digested into smaller products using Ribonuclease (RNase) T1. These digestion products were then separated using two different LC columns and analyzed on a linear ion trap mass spectrometer. One hundred and five products with signature mass values corresponding to specific tRNAs were attempted to be detected and verified by their sequencing information. Of the 105 possible signature digestion products for E.coli total tRNA after digestion with the T1 enzyme 90 were able to be identified but only 40 were able to be confirmed with their CID fragmentation with the use of UPLC separation. Only 73 products were able to be identified using HPLC and 37 of these products were able to be confirmed by their CID fragmentation.
Keywords: UPLC, Mass spectrometry
Abstract:
Based on a large-scale dataset we generated and analyzed, 18 Arabidopsis genes have been identified to have at least one Alternative PolyAdenylation (APA) site at either end of a potential miRNA target in the pre-mRNA and thus generating two transcript isoforms. The use of the APA site (closer to 5’UTR) in the pre-mRNA would eliminate the miRNA target and thus escape miRNA based regulations, while the use of the other APA site (away from 5’UTR) would retain the miRNA target and thus would subject to regulation. We hypothesize that the selection of an APA site itself is a critical regulatory event through which the expression of the gene is modulated by respective miRNA. Another interlinked hypothesis is that miRNA also affect the selection of APA sites by interacting with cleavage and polyadenylation process. Four genes out of the 18 were selected. APA sites and the targets of the miRNA are first confirmed by reverse transcription PCR and over-expression approaches. Then, respective conditions that may lead to differential use of the APA sites of each of the four genes, including developmental and environmental responses, will be used to test if the impact of the APA and miRNA in these responses. Adjacent APA sites of two of those candidate genes have been confirmed. Further results will be presented.
References:
Loke J C, Stahlberg E A, Strenski D G. (2005). Compilation of mRNA Polyadenylation Signals in Arabidopsis Revealed a New Signal Element and Potential Secondary Structures. Plant Physiology. 138: 1457–1468.
Lutz CS. (2008). Alternative polyadenylation: a twist on mRNA 3\' end formation. ACS Chem Biol 3: 609-617.
Mayr, C., and Bartel, D.P. (2009). Widespread Shortening of 3 \' UTRs by Alternative Cleavage and Polyadenylation Activates Oncogenes in Cancer Cells. Cell 138, 673-684.
Rubio-Somoza, I., Cuperus, J., Weigel, D. , Carrington J. C. (2009). Regulation and functional specialization of small RNA–target nodes during plant development. Plant Biology 2009, 12:622–627.
Shen, Y. Genome wide studies of mRNA 3’-end processing signals and alternative polyadenylation in plants, Ph.D. Dissertation, Miami University, Oxford, USA. 2009.
Keywords: messenger RNA, Alternative Polyadenylation, micro RNA
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
In nuclear pre-mRNA splicing, introns are removed through the work of small nuclear RNAs (snRNAs). In the human genome there are two types of introns. Major class introns, the U2-dependent type, which are spliced by U1, U2, U4, U5 and U6 snRNAs, and the minor class introns, the U12 dependent type, which are spliced by U11, U12, U4atac, U5, and U6atac snRNAs. It has been shown that over expression of minor class spliceosomal snRNAs can have an inhibitory effect on the splicing of major class introns. To further test the concept we targeted the HER-2/Neu proto-oncogene, which is over-expressed in 20-30% of breast tumors. We constructed a series of mutant human U6atac and U11 snRNAs to target introns 1, 2, 6, 8, 12 and 13 of HER-2/Neu to block nuclear pre-mRNA splicing and down regulate protein synthesis. We transfected mutant snRNAs in breast cancer cell line MDA-MB-453 to determine the efficacy of our approach on HER-2/Neu pre-mRNA splicing interference. Our preliminary data indicates variable effect on the HER-2/Neu pre-mRNA splicing and protein synthesis in cultured cells. Further experiments are in progress to confirm the efficacy of mutant snRNAs in blockading of HER-2/Neu expression.
Keywords: Breast Cancer, HER-2, U11 and U6atac
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
HuR is an RNA binding protein that plays a protective role during cellular stress. The aims of our present study are to evaluate the regulation of HuR expression and mechanisms of transcriptional control. Previous studies in our lab demonstrated that HuR is expressed in two different forms, one of which contains an approximately 20 base 5’UTR sequence, and one that contains a 150 base G+C rich 5’UTR that is inhibitory to translation. Recovery of renal epithelia from cellular stress induced increased expression of the shorter, more translatable transcript and decreased expression of the longer form. Analysis of HuR upstream regions revealed regulatory sequences for the shorter mRNA. Within the long G+C rich 5’UTR exists multiple copies of the alternate Smad 1/5/8 binding motif, GCCGnCGC. During recovery following the stress ATP depletion, Smad 1/5/8 levels increased. The ability of these Smads to bind relevant motifs in the 5’UTR was confirmed by ChIP and gel shift assays. Transfection of low amounts of exogenous Smad1 increased HuR mRNA expression. Our preliminary data with transcriptional co-activators of Smads such as Sp1 and Sp3 revealed that they are involved in a regulatory role of HuR expression in renal proximal tubule cells. We also demonstrate that the long AU-rich 3’UTR of HuR contains potential regulatory sequences controlling stability of HuR mRNA. Further studies involving auto-regulation and possible micro RNA target binding sites at the level of 3’UTR need to be evaluated. These data suggest that HuR, an RNA-binding protein, is under strong transcriptional regulation and elucidating these mechanisms will help us understand its protective role in renal ischemia- reperfusion injury, ischemic pre-conditioning and other models of cellular stress.
Keywords: HuR, RNA binding proteins, Ischemia
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:
Ribosome assembly requires 180 assembly factor proteins not found in mature ribosomes. These assembly factors are thought to enter and exit the ribosome biogenesis pathway at various times, but a largely unanswered question is what drives the ordered assembly of ribosomes. One hypothesis is that ATPases regulate this process using nucleotide hydrolysis to cause structural rearrangements within the preribosome, driving maturation forward. To investigate the mechanisms that govern ordered assembly, we are studying the DEAD-box protein Drs1 in Saccharomyces cerevisiae. We believe that Drs1, through ATP hydrolysis and helicase activity, functions by either removing early assembly factors from preribosomes or causing a structural rearrangement, to create an environment in which later assembly factors can bind, thus regulating the timing of ribosome biogenesis. We show that Drs1 is required for three steps of pre-rRNA processing. Depletion of Drs1 blocks processing of 27SA2 and 27SA3 pre-rRNAs and traps preribosomes in the nucleolus. Partial loss of function mutants however block processing of 27SB pre-rRNA. While it is known that some assembly factors enter and exit the assembly pathway early and others enter and exit late, we show that depletion of Drs1 causes the formation of a preribosome containing both “early” and “late” assembly factors. In the absence of Drs1, “late” assembly factors copurify with increased amounts of “early” assembly factors and pre-rRNA intermediates. One model is that depletion of Drs1 causes “late” assembly factors to enter the nucleolus and associate with preribosomes prematurely. Another scenario is that preribosomes are very dynamic early in assembly, possibly a result of ATP hydrolysis by Drs1 and other ATPases, causing “late” proteins to be weakly associated with preribosomes. Depletion of Drs1 could cause these early particles to become less dynamic and more stable, therefore allowing detection of these “late” proteins.
References:
Henras, A.K., Soudet, J., Gerus, M., Lebaron, S., Caizergues-Ferrer, M., Mougin, A., and Y. Henry. 2008. The post-transcriptional steps of eukaryotic ribosome biogenesis. Cell. Mol. Life. Sci. 65:2334-2359.
Ripmaster, T.L., G.P. Vaughn, and J.L. Woolford, Jr. 1992. A putative ATP-dependent RNA helicase involved in Saccharomyces cerevisiae ribosome assembly. Proc. Natl. Acad. Sci. USA 89:11131-11135.
Adams, C.C., J. Jakovljevic, J. Roman, P. Harnpicharnchai, and J.L. Woolford, Jr. 2002. Saccharomyces cerevisiae nucleolar protein Nop7 is necessary for biogenesis of 60S ribosomal subunits. RNA 8:150-165.
Keywords: Ribosome, ATPase, preribosomal RNA processing
Abstract not available online - please check the printed booklet.
Abstract:
Proximal spinal muscular atrophy (SMA) is a neuromuscular disease caused by low levels of SMN protein due to loss of the Survival Motor Neuron-1 (SMN1) gene. Humans have a duplicate gene, SMN2, that generates low levels of SMN protein due to a C>T point mutation that skips exon 7. We generated a mouse model that recapitulates SMN2 altered splicing by engineering the exon 7 C>T mutation into the homologous mouse Smn gene. The C>T mutation in Smn induces skipping of exon 7 in multiple tissues in the mouse, reduces SMN protein levels, and leads to a mild form of SMA exhibited by reduced hind limb grip strength, rearing, and activity. Increasing SMN expression by correcting splicing or increasing transcription of SMN2 are attractive therapeutic options in SMA patients. However, the timing of SMN replacement will be crucial in the treatment of SMA. Studies in zebrafish predict SMN function during embryogenesis may be important for axonal pathfinding, while mouse models and SMA disease progression in humans suggest that post-natal treatment may be sufficient to protect motor neurons. AAV9 encoding SMN has shown that early postnatal SMN expression in spinal cord motor neurons is sufficient to rescue survival though complete rescue of normal development was not observed, indicating that earlier treatment may be required for full restoration of SMN function and disease correction. To address the question of optimal therapeutic timing for SMA we have developed a temporally inducible transgenic mouse in which the expression of human SMN cDNA is under the control of tamoxifen inducible Cre-recombinase. In our inducible mouse, SMN expression after tamoxifen treatment was validated in adult mice and PND1.5 treated neonatal mice. The temporal induction of SMN expression was further validated in these mice in various tissues including the brain and spinal cord. When crossed to SMA mouse models our inducible SMN mouse model will allow for the temporal induction of SMN at varying time-points during development, both in utero and postnatally, to evaluate the time-point in which SMN replacement is required for SMA correction. The therapeutic window determined from these experiments can then be used in our Smn C>T mouse model to validate the use of drug therapies targeting splicing correction in the treatment of SMA mice, and in turn SMA patients.
Keywords: Splicing, Spinal Muscular Atrophy, Mouse Model
Abstract:
Aminoacyl-tRNA synthetases are responsible for charging the correct amino acid onto cognate tRNAs. To ensure fidelity in protein synthesis, a sub-set of these enzymes have evolved editing mechanisms. “Pre-transfer” editing refers to hydrolysis of the misactivated aminoacyl-adenylate intermediate, whereas “post-transfer” editing is hydrolysis of the mischarged tRNA. In most bacterial prolyl-tRNA synthetase (ProRS) systems, an editing domain known as INS is responsible for post-transfer editing of mis-charged Ala-tRNAPro. However, a sub-set of ProRSs have a truncated “mini–INS” that is unlikely to support catalytic activity. To explore the function of the mini-INS and to better understand why a full-length INS is not needed in some bacteria, we chose to investigate Rhodopseudmonas palustris (Rp) ProRS. The mini-INS consists of 28 residues forming 2 α-helices and a loop. We showed that RpProRS activates Cys, Pro, and Ala with 0-, 5-, and 25-fold reduced catalytic efficiency relative to EcProRS. The estimated KM of 1.0 M for Ala is significantly higher than the concentration of Ala in the cell. In addition, RpProRS shows pre-transfer activity against Ala that is comparable to that of EcProRS. Therefore, based on the lower overall activation of Ala and the relatively robust pre-transfer editing activity, we conclude that a full-length INS domain for post-transfer editing is not needed in the Rp system.
Based on the known dimeric structure of RpProRS and preliminary gel filtration studies, we hypothesize that residues in the mini-INS domain have been maintained to facilitate tight dimer formation. Modeling studies suggest that acidic residues on the loop in the mini-INS domain interact with basic residues proximal to the known dimerization interface. We hypothesize that this salt bridge aids in the oligomerization process. In support of this hypothesis, all mini-INS deletion variants prepared to date were found to be misfolded and formed inclusion bodies. Taken together, our results suggest that the mini-INS is critical for preserving RpProRS’s active dimeric structure.
Keywords: Rhodopseudomonas palustris, aminoacyl-tRNA synthetases, post-transfer editing
Abstract not available online - please check the printed booklet.
Abstract:
Multiple peptide resistance factor (MprF) is a protein that transfers amino acids from aminoacyl-tRNAs to phosphatidylglycerol (PG) in the cytoplasmic membrane. Aminoacylation of PG is a strategy utilized by bacteria to decrease the net negative charge of the cell envelope, thereby diminishing affinity for positively charged molecules and allowing for adaptation to environmental changes. Previous studies of MprF with specificity for the amino acid lysine have shown it to be an important virulence factor of Staphylococcus aureus, providing resistance to both host cationic antimicrobial peptides and therapeutic antibiotics. The physiological role of the highly diverse mprF homologs in other bacterial species remains largely unstudied. Examination of the physiological activity of MprF in Listeria monocytogenes and Bacillus subtilis by Biolog phenotypic microarray (PM) revealed phenotypes correlating with direct modulation of cell envelope charge and a new class of phenotypes that indicate a global effect on membrane properties. These two classes of phenotypes are consistent between the two organisms studied. Implicating MprF as a promising target for future development of antimicrobial therapies.
References:
1. Peschel, A., et al., Staphylococcus aureus resistance to human defensins and evasion of neutrophil killing via the novel virulence factor MprF is based on modification of membrane lipids with l-lysine. J Exp Med, 2001. 193(9): p. 1067-76.
2. Thedieck, K., et al., The MprF protein is required for lysinylation of phospholipids in listerial membranes and confers resistance to cationic antimicrobial peptides (CAMPs) on Listeria monocytogenes. Mol Microbiol, 2006. 62(5): p. 1325-39.
3. Staubitz, P. and A. Peschel, MprF-mediated lysinylation of phospholipids in Bacillus subtilis--protection against bacteriocins in terrestrial habitats? Microbiology, 2002. 148(Pt 11): p. 3331-2.
4.Roy, H., Tuning the properties of the bacterial membrane with aminoacylated phosphatidylglycerol. IUBMB Life, 2009. 61(10): p. 940-53.
Keywords: lipid modification, aminoacylphosphatidylglycerol synthase, tRNA
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
Thirty-one RNA duplexes containing single nucleotide bulge loops were optically melted in 1M NaCl; and, the thermodynamic parameters ÄH o, ÄSo, ÄGo37, and Tm for each sequence were determined. The bulge loops were of the group II variety where the bulged nucleotide is identical to one of its nearest-neighbors leading to ambiguity as to the exact position of the bulge. Sequences from this study were combined with sequences from previous studies (Znosko et al., (2002) Biochemistry 41, 10406-10417), thus examining all possible group II single nucleotide bulge loop sequences. The combined data were used to develop a model to predict the free energy of an RNA duplex containing a single nucleotide bulge. As observed previously for group I bulge loops (Blose et al., (2007) Biochemistry 46, 15123-15135), neither the identity of the bulge nor the nearest-neighbor base pairs had an effect on the influence of the bulge loop on duplex stability. The destabilization of the duplex by the bulge was primarily related to the stability of the stems adjacent to the bulge. Specifically, there was a direct correlation between the destabilization of the duplex and the stability of the less stable duplex stem. Since there is an ambiguity of the bulge position for group II bulges, several different stem combinations are possible. The destabilization of group II bulge loops is similar to the destabilization of group I bulge loops, if the second least stable stem is used to predict the influence of the group II bulge. In-line structure probing of the group II bulge loop embedded in a hairpin indicates that the bulged nucleotide is the one positioned further from the hairpin loop.
References:
Blose JM, Manni ML, Klapec KA, Stranger-JonesY, Zyra AC, Sim V, Griffith CA, Long JD, Serra MJ. (2007). Non-nearest-neighbor dependence of the stability for RNA bulge loops based on the complete set of group I single-nucleotide bulge loops. Biochemistry 46, 15123-15135.
Znosko BM, Silvestri SB, Volkman H, Boswell B, Serra MJ. (2002). Thermodynamic parameters for an expanded nearest-neighbor model for the formation of RNA duplexes with single nucleotide bulges. Biochemistry 41, 10406-10417.
Keywords: group II bulge, duplex stability, in-line structure probing
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
Significant improvements were made on a JAVA program designed to search though bpseq files describing secondary structure (1). This program features a graphical user interface and allows the user to search bpseq files for various RNA secondary structural motifs. The search yields information about position and number of times the query appears in the user specified dataset. The improvements include providing a visualization of the query motif(2), and a more thorough exploration of the helical regions including predicted thermodynamics based on the helix sequence. These search results give insight as to what structures and sequences are prevalent in nature. We intend to use the results to gather statistical information about the stems surrounding various RNA secondary structural elements(3).
For instance after a search for all single nucleotide budges the resulting dataset was easily explored. The mean length for stems surrounding a single nucleotide was found to be 4 nucleotides with an average predicted Gibbs free energy of the stem of -6.20 kJ/mol. The average ratio of number of nucleotides in the longer stem to number of nucleotides in the shorter stem is 2.2:1, and the average ratio of the more stable to less stable stem free energy is 5.5:1.
References:
1 Cannone J.J., Subramanian S., Schnare M.N., Collett J.R., D\'Souza
L.M., Du Y., Feng B., Lin N., Madabusi L.V., MÜller K.M., Pande N.,
Shang Z., Yu N., and Gutell R.R. (2002). The Comparative RNA Web (CRW)
Site: An Online Database of Comparative Sequence and Structure
Information for Ribosomal, Intron, and Other RNAs. BioMed Central
Bioinformatics, 3:2.
2 Kévin Darty, Alain Denise, Yann Ponty
VARNA: Interactive drawing and editing of the RNA secondary structure.
Bioinformatics, pp. 1974-1975, Vol. 25, no. 15, 2009
3 Blose JM, Manni ML, Klapec KA, Stranger-Jones Y, Zyra AC, Sim V,
Griffith CA, Long JD, Serra MJ. Non-nearest-neighbor dependence of the
stability for RNA bulge loops based on the complete set of group I
single-nucleotide bulge loops. Biochemistry (2007) 46:15123–15135.
Keywords: BioInformatics, Secondary structure, JAVA search program
Abstract:
Ribosomes have elaborate structures composed of ribosomal proteins embellished in a core of ribosomal RNA (rRNA). We are interested in how these ribonucleoprotein complexes are assembled. Ribosome biogenesis is a tightly controlled process involving a series of intertwined events: processing and modification of the rRNA primary transcript, hierarchical and cooperative assembly of ribosomal proteins, and remodeling of preribosome (pre-rRNP) conformations to ultimately form mature functional ribosomes. We study the molecular mechanisms governing the formation of the large ribosomal subunit. Assembly of the 60S subunit is precisely coordinated - starting in the nucleolus and finishing in the cytoplasm. Each step is mediated by trans-acting assembly factors that transiently associate with preribosomes. Thus far, most studies on ribosome assembly have focused on characterizing how the absence of or mutations in assembly factors affect the assembly pathway. In contrast, although most ribosomal proteins are crucial to the formation of properly functioning ribosomes, very little is known about their role in the biogenesis pathway. We and others have recently tested how in vivo depletion of each ribosomal protein affects ribosome assembly and found that the absence of individual ribosomal proteins leads to defects in distinct pre-rRNA processing steps. Of the thirty-one large subunit ribosomal proteins (rpLs) tested, nine specifically affect processing of 27SA to 27SB pre-rRNA, seven affect processing of 27SB to 7S and 25.5S pre-rRNAs, and eight affect conversion of 7S pre-rRNA to 5.8S mature RNA. Our lab is particularly interested in the 27A3 and 27B pre-rRNA processing steps; thus we started to characterize in further detail the roles of each rpL affecting these events. We investigated the effects of rpL depletion on pre-rRNA processing, association of assembly factors and other rpLs with preribosomes, and export of pre-rRNAs from nucleus to cytoplasm.
References:
Ferreira-Cerca, S., Pöll, G., Kühn, H., Neueder, A., Jakob, S., Tschochner, H., and Milkereit, P. (2007) Analysis of the in vivo assembly pathway of eukaryotic 40S ribosomal proteins. Mol Cell. 28: 446-457.
O’Donohue, M.F., Choesmel, V., Faubladier, M., Fichant, G., and Gleizes, P.E. (2010) Functional dichotomy of ribosomal proteins during the synthesis of mammalian 40S ribosomal subunits. J. Cell. Biol. 190:5 853–866.
Pöll, G., Braun, T., Jakovljevic, J., Neueder, A., Jakob, S., Woolford, J.L., Jr., Tschochner, H., Milkereit, P. (2009) rRNA maturation in yeast cells depleted of large ribosomal subunit proteins. PLoS One. 4:e8249. doi:10.1371/journal.pone.0008249.
Keywords: ribosome assembly, ribosomal proteins, pre-rRNA processing
Abstract:
Transfer RNAs (tRNAs) are primarily responsible for translation of the genetic code and function in numerous other processes such as viral replication, amino acid biosynthesis, and cell wall remodeling. tRNAs have also been shown to regulate gene expression by acting as sensors of the translation status of the cell, and it has been suggested that these highly stable molecules could provide a structural framework for small RNAs (sRNAs) that regulate other cellular processes. A pseudo-tRNA from Bacillus cereus, tRNAOther, does not associate with polysomes, suggesting a role outside translation. Deletion of tRNAOther leads to changes in cell wall morphology and significantly reduces resistance against a number of unrelated antibiotics including vancomycin, puromycin, novobiocin, and rifapentin. The deletion of tRNAOther is also accompanied by significant changes in the expression of numerous genes involved in oxidative stress response, several of which contain significant sequence complementarities to tRNAOther, suggesting a possible regulatory sRNA function. Recent studies have shown that many bactericidal antibiotics act in part by inducing intracellular oxidative stress (1). Additional studies have shown that NO produced by bacterial nitric oxide synthase (NOS) can act as an antioxidant, thereby reducing oxidative stress (2). Deletion of tRNAOther significantly reduces nos transcript levels and intracellular NO production. Expression of tRNAOther changes in response to extracellular iron concentration, consistent with the presence of three putative ferric uptake regulator (Fur) bindings sites in the predicted promoter region of the corresponding gene. Unbound intracellular iron is involved in oxidative stress, in that ferrous iron can generate hydroxyl radicals through the Fenton reaction. Taken together, these findings indicate that tRNAOther is an iron-responsive sRNA that modulates antibiotic resistance by regulating the expression of multiple targets involved in oxidative stress response.
References:
1. Kohanski, M.A., et al. A common mechanism of cellular death induced by bactericidal antibiotics. 2007. Cell. 130:797-810.
2. Gusarov, I., et al. Endogenous nitric oxide protects bacteria against a wide spectrum of antibiotics. 2009. Science. 325:1380-1384.
Keywords: pseudo-tRNA, sRNA, antibiotic resistance
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:
Studies of the yeast tRNA methyltransferase Trm4p revealed the partial reversibility of RNA m5C methyltransferases that function with a dual-cysteine mechanism. Trm4p forms covalent complexes with methylated tRNA in the presence of S-adenosylhomocysteine and complex formation increases as the pH becomes more acidic. We anticipate that YebU (RsmF), a bacterial m5C methyltransferase is also partially reversible. YebU modifies cytosine 1407 in 16S rRNA, but only in the context of an intact 30S subunit. A YebU-30S complex would preclude association of the 30S and 50S ribosomal subunits, resulting in blocked translation. Although this pH based translational inhibition is theoretical, it could be significant in strongly acidic environments, thereby influencing acid survival of YebU expressing bacteria like E. coli and Salmonella.
Here we provide evidence that YebU has a role in acid survival. The growth of E. coli strain CP79ÄyebU was compared to the growth of the parental CP79 strain in buffered rich media at various pH values. Multiple experiments revealed a substantial growth difference between the mutant and wild type strains at pH values just above the acid limit for growth. The difference was observed when either stationary or log phase cells were shifted to low pH, but the effects were stronger and longer lasting with log phase cells. Plating experiments with log phase cells demonstrated only 5% survival of the CP79ÄyebU cells after 24 hours at pH 4.6. Growth studies with the YebU mutant from the Keio knockout collection showed similar growth deficiencies relative to the corresponding parental strain, therefore the results obtained are not specific to the CP79 strain. The results have encouraged us to seek biochemical evidence for translational regulation by YebU and to further pursue the general concept that RNA m5C methyltransferases may function as acid sensors in biological systems.
Keywords: m5C, Acid Sensor, RNA Methyltransferase
Abstract:
The Ribosomal Databse Project: Tools and sequences for rRNA analysis.
Benli Chai, Jordan A. Fish, Qiong Wang, James M. Tiedje and James R. Cole.
Center for Microbial Ecology, Michigan State University, 2225A Biomedical Physical Sciences, East Lansing, MI
The ribosomal small subunit rRNA (16S) gene is the most frequently used molecular marker
for studying microorganisms. The Ribosomal Database Project (RDP; http://rdp.cme.msu.edu) offers aligned and annotated 16S rRNA sequence data and analysis services to the research community. Updated monthly, RDP maintained 1,418,497 aligned and annotated quality-controlled public rRNA sequences as of September 2010, while researchers maintain over 5 million sequences in their myRDP accounts. RDP products are widely used in molecular phylogeny and evolutionary biology, microbial ecology, bacterial identification, characterizing microbial populations, and in understanding the diversity of life.
While Current-Generation sequencing technologies such as 454 and Illumina are transforming studies of microbial diversity by offering greatly extended sequencing breadth and depth, they also bring in challenges in data processing and analysis. The RDP Pyrosequencing Pipeline provides an effective toolkit to automate the computationally-intensive processing and analysis of these large datasets. The Pipeline takes raw high-throughput reads through initial processing steps to hierarchical clustering into OTUs or taxonomic assignment using the RDP Classifier. Several common ecological metrics are calculated including Chao1, Shannon Index and rarefaction. Results are available in formats suitable for common statistical and ecological packages. Other RDP tools include the RDP Classifier, also available as an open-source package; Sequence Match for finding nearest neighbors; Library Compare for determining differentially represented taxa between two libraries; RDP Probe Match for determining taxonomic coverage of primers and probes; Tree Builder for rapid phylogenetic tree construction; and Browsers that provide entry to the public sequences.
Keywords: rRNA, pyrosequencing, bioinformatics
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 recent large scale genomic analyses have indicated that long non-coding RNAs constitute a major group of regulatory factors in higher eukaryotes. However, due to their novelty, very few studies have addressed their cellular function and their mechanistic strategies in sufficient detail. In our studies, we have focused on the BORG RNA, a ~2700 nucleotide long message which is induced in response to the activation of BMP2 signaling pathway. BORG is a spliced and polyadenylated RNA, however, it is localized to the nuclei. Tissue expression analyses have indicated that in the mouse, BORG has a very restricted expression pattern, with its expression being detectable only in neural tissues and kidneys. Analysis of the function of BORG in the N2A neuroblastoma cells, a commonly used neuronal differentiation model, indicated that BORG is required for differentiation in these cells. While shRNA-mediated knockdown of BORG blocked neuronal differentiation in N2A cells, BORG overexpression resulted in faster and more efficient morphological differentiation in these cells. In order to define the role of BORG in differentiation of other cell types, we determined the effect of its overexpression in the C2C12 myoblasts and C3H10T1/2 fibroblastic cells. Interestingly, BORG overexpression resulted in reprogramming of both these cell lines into neurons. In order to gain insight into the mechanism of this RNA-mediated reprogramming, we have launched a comprehensive study of the factors which interact with this non-coding RNA. Preliminary results from this study will be presented.
Keywords: long non-coding RNAs, BORG, reprogramming
Abstract not available online - please check the printed booklet.
Abstract:
The GAIT (Gamma-interferon Activated Inhibitor of Translation) complex inhibits translation of inflammatory transcripts in human myeloid cells, thereby preventing excessive accumulation of potentially harmful proteins. During the inhibition phase we observed a low-level “trickle” of translation of GAIT target mRNAs independent of mRNA amount. In circumstances where inflammatory proteins also have protective roles, e.g., vascular endothelial growth factor-A (VEGF-A), complete knock-down can cause tissue injury and tumor growth. Thus, incomplete inhibition of gene expression by the GAIT system might be beneficial. We discovered a novel mechanism that compels low-level translation of GAIT target mRNAs despite robust inhibition by the GAIT system. Glutamyl-prolyl tRNA synthetase (EPRS) is the sole component of the GAIT complex that binds the GAIT element-bearing mRNAs. We found a truncated form of EPRS, termed \
Keywords: alternative polyadenylation, translational trickle, VEGF-A
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:
Both DNA and RNA can serve as powerful building blocks for bottom-up fabrication of nanostructures. A pioneering concept proposed by Ned Seeman 30 years ago has led to an explosion of knowledge in DNA nanotechnology (1). RNA can be manipulated with simplicity characteristic of DNA, while possessing noncanonical base-pairing, versatile function and catalytic activity similar to proteins (2). However, standing in awe of the sensitivity of RNA to RNase degradation has made many scientists flinched from RNA nanotechnology. In this context, we will report the construction of stable RNA nanoparticles resistant to RNase digestion. The chemically modified RNA retained its property for correct folding in dimer formation, appropriate structure in procapsid binding, and biological activity in gearing phi29 nanomotor to package viral DNA and producing infectious viral particles. Our results demonstrate that it is practical to produce RNase resistant, biologically active and stable RNA for application in nanotechnology (3).
References:
(1) Seeman, N. C. Nanomaterials Based on DNA, Annu. Rev. Biochem. 2010, 79, 65-87
(2) Guo P., Coban O., Snead NM, Trebley J, Hoeprich S, Guo S, Shu Y, Engineering RNA for targeted siRNA delivery and medical application, Adv. Drug Deliv. Rev. 2010, 62, 650-66.
(3) Liu J, Guo S, Cinier M, Shu Y, Chen C, Shen G, and Guo P, Fabrication of Stable and RNase-Resistant RNA Nanoparticles Active in Gearing the Nanomotors for Viral DNA-Packaging, submitted.
Keywords: 2-F modification, RNase resistant nanoparticle, phi29 DNA-packaging nanomotor
Abstract:
RNA base triples are clusters of three nucleotides interacting edge-to-edge through hydrogen bonding. To understand the sequence constraints imposed by base triples, we have introduced a classification that accounts for all observed triples and predicts triples not yet observed in the 3D structure database.
Each triple family belongs to two \
References:
Chen, V.B., I.W. Davis, and D.C. RIchardson (2009). KiNG (Kinemage, Next Generation): a versatile interactive molecular and scientific visualization program. Protein Sci. 18:2403-9.
Sarver, M., Zirbel, C.L., Stombaugh, J., Mokdad, A., & Leontis, N. (2008). FR3D: Finding local and composite recurrent structural motifs in RNA 3D structures. Journal of Mathematical Biology, 56(1), 215-252.
Stombaugh, J., C.L. Zirbel, E. Westhof, and N.B. Leontis (2009). Frequency and isostericity of RNA base pairs. Nucleic Acids Res. 37:2294-312.
Word, J. M., Lovell, S. C., LaBean, T. H., Taylor, H. C., Zalis, M. E., Presley, B. K., et al. (1999). Visualizing and quantifying molecular goodness-of-fit: Small-probe contact dots with explicit hydrogen atoms. Journal of Molecular Biology 285:1711-1733.
Keywords: RNA base triple, Superfamily, Serious steric clashes
Abstract:
Regulation of cell volume is a fundamental process in living cells. Cells have developed adaptive mechanisms to respond to physiological changes in extracellular osmolarity (as known for kidney medulla cells) and restore cell volume. However, pathological exposure of cells to increased osmolarity (inflammation, diabetes, hypernatremia) induces a competing to adaptation apoptotic program. Oxidative stress is part of the pathologic features of hypertonic stress. The cellular response to hypertonic stress involves global decrease of protein synthesis, in a manner independent of eIF2a phosphorylation, the master regulator of most stress responses. Prior studies with Mouse Embryonic Fibroblast (MEF) cells in our lab have shown that osmotic stress triggers eIF2α phosphorylation, but it is not the cause of translational repression. MEFs deficient in eIF2α phosphorylation showed a dramatic decrease in protein synthesis when subjected to hyperosmolar treatment (Bevilaqua et. al., 2010). We are interested to investigate the mechanism of the eIF2a phosphorylation independent translational repression. A study reported in the literature, showed that oxidative stress induces tRNA cleavage in mammalian cells in a manner independent of phophorylation of eIF2α (Yamasaki et. al. 2009). We propose to study the involvement of this mechanism during hypertonic stress. This project aims to identify/quantify specific populations of cleaved tRNA fragments in MEFs under hypertonic stress using a microarray based technique. Selectivity if observed, may point to cleaved tRNA fragments being regulatory molecules in the osmotic stress signaling pathway. In addition we are also exploring the mechanism of translational repression caused by these tRNA fragments and the mechanism of tRNA substrate recognition for cleavage by specific nucleases.
References:
Bevilacqua E, Wang X, Majumder M, Gaccioli F, Yuan CL, Wang C, Zhu X, Jordan LE, Scheuner D, Kaufman RJ, Koromilas AE, Snider MD, Holcik M, Hatzoglou M (2010). eIF2alpha phosphorylation tips the balance to apoptosis during osmotic stress. J Biol Chem.;285(22):17098-111.
Yamasaki S, Ivanov P, Hu GF, Anderson P. (2009) Angiogenin cleaves tRNA and promotes stress-induced translational repression. J Cell Biol.;185(1):35-42.
Keywords: tRNA Cleavage, Hyperosmotic stress
Abstract:
Transition state theory defines catalysis as the preferential stabilization of a transition state relative to the ground state. Thus, the proficiency of an enzyme is directly related to its ability to recognize and stabilize the transition state. Characterizing the geometry, bonding, and charge distribution of the transition state is therefore critical for understanding catalysis. Despite decades of research on the mechanisms of ribozyme and protein catalysis of RNA strand cleavage by 2’-O-transphosphorylation, there remain significant ambiguities regarding the specific catalytic modes that are used and the roles of active site residues. To help resolve these issues, we are investigating the interactions of enzymatic and non-enzymatic catalysts with the transition state using kinetic isotope effects. The effects on reaction rate of substituting atoms undergoing reaction with heavier, stable isotopes provides information on transition state structure and the interactions with catalysts. We have developed new methods to synthesize RNAs containing site specific isotopic substitutions and for determining the effects of isotopic substitution on reaction rate constants. KIEs for RNA 2’-O-transphosphorylation are providing new insights and the necessary mechanistic clarity for understanding enzyme catalysis. Results to date demonstrate that catalysis by hydroxide occurs by nucleophilic activation by specific base catalysis. KIEs for acid catalysis support modes in which this reaction proceeds by a stepwise mechanism with a phosphorane intermediate, and in which the protonation of a non-bridging phosphoryl oxygen helps to stabilize the intermediate. Results from metal ions catalyzed transphosphorylation reactions indicate that catalysis occurs by leaving group stabilization thorough direct coordination, or by general acid catalysis. Results for RNase A demonstrate a concerted, rather than stepwise mechanism and are consistent with interactions with both the nucleophile and leaving group, but suggest that leaving group bonding is influenced by active site protonation. Together the results demonstrate the range of mechanisms and transition states that are possible for RNA reactions and reveal how catalytic interactions influence transition state bonding.
References:
Harris ME, Dai Q, Gu H, Kellerman DL, Piccirilli JA, Anderson VE. Kinetic isotope effects for RNA cleavage by 2\'-O- transphosphorylation: nucleophilic activation by specific base. J Am Chem Soc. 132(33):11613-21 (2010).
Dai Q, Frederiksen JK, Anderson VE, Harris ME, Piccirilli JA. Efficient synthesis of [2\'-18O]uridine and its incorporation into oligonucleotides: a new tool for mechanistic study of nucleotidyl transfer reactions by isotope effect analysis. J Org Chem. 73(1):309-11 (2008).
Keywords: kinetic isotope effect
Abstract:
The RNA processing exosome was originally defined as an evolutionarily conserved multi-subunit complex of ribonucleases responsible for the processing and/or turnover of stable RNAs and mRNAs. Results from several in vivo systems challenge the prevailing model of exosome complex function, and we have proposed the exozyme model to address the discrepancies. The exozyme model posits that, in addition to assembling into the core, exosome subunits compose smaller complexes with distinct roles in RNA metabolic events. Here, we test the exozyme model by using RNAi to deplete exosome subunits and an exosome cofactor in Drosophila melanogaster S2 tissue culture cells and assay the effects on hsp70 and hsp26 mRNA levels using S1 nuclease protection assays. As predicted by the exozyme hypothesis, we find different effects on hsp70 and hsp26 mRNA levels when different subunits are depleted. We also observe continued accumulation of heat shock transcripts when we deplete either Rrp4 or Rrp40. This study provides evidence for multiple independent functions for individual exosome subunits and intimates greater complexity of exosome subunit assemblages than heretofore anticipated.
Keywords: exosome, Rrp4, Rrp40
Abstract:
ABSTRACT
Exosome complexes are composed of 10 to 11 subunits and are involved in multiple facets of 3’to 5’ RNA processing and turnover. The current paradigm stipulates that a uniform, stoichiometric core exosome, composed of single copies of each subunit, carries out all RNA metabolic functions attributed to the complex; however, available data raises questions about whether individual subunits contribute to RNA metabolic functions exclusively within the complex. We have proposed the exozyme hypothesis, a novel model stipulating that subsets of exosome subunits and cofactors assemble into a continuum of compositionally distinct complexes with different RNA specificities to resolve this discrepancy. Here, we test the predictions of the exozyme model via bioinformatic analysis of microarray data obtained from exosome subunit depleted cells. The transcriptomic analyses show individual depletion of individual exosome subunits contributes differentially to mRNA levels. Extending upon these findings will advance our understanding of the underlying mechanisms regulating RNA processing and turnover.
Keywords: Exosome, Exozyme, microarray
Abstract:
Eukaryotes monitor mRNA transcript form and quality with different surveillance systems. One such system, called nonsense-mediated mRNA decay (NMD), can detect and help eliminate transcripts with premature termination codons (PTCs). The core components of the NMD pathway were first characterized in Saccharomyces cerevisiae and orthologs have since been identified in all other eukaryotes examined (Chang, 2007; Maquat, 2004). While the molecular mechanisms that underlie the selective degradation of PTC-containing transcripts are increasingly well understood, less is known about the source or nature of NMD substrates in wild-type cells. Various microarray and bioinformatics-based studies in S. cerevisiae, C. elegans, and mammalian cell culture suggest that PTC-containing products of alternative splicing may be an important class of physiologic substrate of NMD (Lewis, 2003; He, 2003; Wittmann, 2006). The recent identification and characterization of core NMD-factors in Schizosaccharomyces pombe have allowed us to undertake a systematic, microarray-based screen for S. pombe physiologic targets of NMD (Rodriguez-Gabriel, 2006). Using PCR-microarrays from the Stony Brook Spotted Microarray Facility, we have identified more than 100 transcripts at least two-fold more abundant in an NMD(-) background, compared to wild-type. The large number of intron-containing genes in the S. pombe genome (~43% compared to ~5% in S. cerevisiae) suggests that the pool of natural targets identified by our arrays might include alternatively spliced pre-mRNAs. We are currently using RT-PCR with primers flanking predicted splice junctions of each transcript in order to identify these putative NMD-targeted splice products. Studies such as this one will help us understand the role NMD plays in post-transcriptional regulation of gene expression.
References:
1. Chang YF, et al. Annu Rev Biochem. 2007; 76: 51-74
2. Maquat LE, Curr Genomics. 2004; 5: 175–190
3. Lewis BP, PNAS. 2003; 100(1): 189–192
4. He F, et al. Mol Cell. 2003; 12(6):1439-1452
5. Wittmann J, et al. Mol Cell Biol. 2006; 26(4): 1272-1287
6. Rodriguez-Gabriel MA, et al., MCB. 2006; 26(17): 6347–6356
Keywords: NMD, mRNA surveillance, yeast
Abstract not available online - please check the printed booklet.
Abstract:
Gene expression is regulated at many different levels, including post-transcriptionally. One such regulatory mechanism involves nonsense-mediated decay (NMD), a cellular quality control pathway that selectively degrades mRNAs with premature termination codons (PTCs). NMD targets two populations of transcripts: 1) abnormal mRNAs that arise due to mutations or processing errors; and 2) physiological targets—those mRNAs that are targeted in wild type cells as part of normal regulation of gene expression (Neu-Yilik and Kulozik, 2008). One such target is Aly-3, a nuclear export factor found in the nematode C. elegans. Primarily nuclear, Aly-3 binds to RNA transcripts and transports them from the nucleus to the cytoplasm. Using microarrays, RT-PCR, and qRT-PCR, we showed aly-3 to be alternatively spliced. Our results demonstrate the presence of an alternative splice form of aly-3 in worms that could not perform NMD. This isoform is absent in wild-type animals, suggesting that NMD participates in the regulation of the pool of aly-3 transcripts. There are a number of testable models that explain the functional utility of this kind of NMD-dependent splicing. For example, Aly-3 may control its own levels through a homeostatic feedback loop in which the Aly-3 protein drives spicing in an unproductive direction. Such a loop plays a role in the expression of ribosomal protein L12 and the splicing factor SC35, natural targets of mRNA surveillance in C. elegans (Mitrovich and Anderson, 2000; Sureau et al., 2001). Regulatory patterns such as these are consistent with current models that propose widespread coupling of NMD and alternative splicing (Lareau et al., 2007). Alternatively, there may be certain cellular or environmental conditions that lead to NMD-dependent splicing of aly-3 (and others). Predictions made by these different models are currently being tested.
References:
Lareau LF, Brooks AN, Soergel DA, Meng Q, and Brenner SE. The coupling of alternative splicing and nonsense-mediated mRNA decay. Adv Exp Med Biol. 2007; 623:190-211.
Mitrovich QM and Anderson P. Unproductively spliced ribosomal protein mRNAs are natural targets of mRNA surveillance in C. elegans. Genes Dev. 2000; 14(17):2173-84.
Neu-Yilik G and Kulozik AE. NMD: multitasking between mRNA surveillance and modulation of gene expression. Adv Genet. 2008; 62:185-243.
Sureau A, Gattoni R, Dooghe Y, Stévenin J, Soret J. SC35 autoregulates its expression by promoting splicing events that destabilize its mRNAs. EMBO J. 2001; 20(7):1785-96.
Keywords: NMD, alternative splicing, C elegans
Abstract not available online - please check the printed booklet.
Abstract:
We have previously shown that two of the spliceosomal snRNAs, U6 and U2, form a basepaired complex that can catalyze a two step splicing reaction on short RNA substrates (Valadkhan et al., 2009). This reaction not only resembles group II intron splicing and spliceosomal catalysis in its chemistry, but also occurrs in the vicinity of an invariant sequence in U6 which is known to be close to the 5' splice site during the catalysis of the first step of spliceosomal splicing (Lee et al., 2010). Mutations in this invariant sequence that block spliceosomal splicing are also incompatible with the snRNA-mediated catalysis. Further, we have shown that an intramolecular stemloop (ISL) in U6, which closely resembles the catalytically critical domain V of group II introns, is required for the snRNA-mediated splicing.
Numerous mechanistic and structural similarities between the spliceosomal snRNAs and group II introns have led to the hypothesis that the snRNAs are descendants of group II-like introns. Recent structural studies have elucidated the presence of a base triplet interaction in the catalytic core of group II introns (Toor et al., 2008). This interaction, which formed between the base of domain V and the linker sequence between domains II and III in group II introns (J2/3), led to the juxtaposition of catalytically essential residues. Interestingly, J2/3 is thought to be the functional counterpart of the evolutionarily invariant ACAGAGA sequence of U6. To determine whether an analogous interaction is formed in the folded structure of the catalytically active U6/U2 complex, we have made mutations at the base of the ISL and in the ACAGAGA sequence and experiments are under way to determine the catalytic activity of the resulting constructs.
Keywords: snRNA, Catalysis, Group II introns
Abstract:
Ribosomal protein (rp) S5 belongs to the family of ribosomal proteins that contains rpS7 from prokaryotes and rpS5 from eukaryotes. RpS5 forms part of the exit (E) site on the 40S ribosomal subunit and is essential for cell viability of yeast. To investigate the function of the rpS5 and in particular the role of the N-terminal extension of the yeast protein (absent in bacteria), we obtained and characterized yeast strains in which the wt yeast rpS5 was replaced by its truncated variants, lacking 13, 24, 30 and 46 N-terminal amino acids, respectively. Biochemical analysis of the mutant yeast strains previously showed that the N-terminal part of the yeast S5 plays important roles in ensuring the efficiency and accuracy of elongation and initiation processes. In particular, we have found that cap-dependent initiation in YS5-46 strain (lacking 46 N-terminal amino acids) was reduced by about 50% as compared to the wild type strain. Here we further show that this strain fails to support re-initiation as evident by the use of GCN4-lacZ reporter constructs. Comparison of lacZ expression from various GCN4-lacZ reporter constructs (containing all four uORFs and/or two uORFs (uORFs 1&4) as well as one uORF4) allowed us to suggest that YS5-46 yeast strain possesses an initiation defect downstream of the 48S complex assembly. An increased association of initiation factors eIF2alpha, eIF1 and eIF5B with mutant 40S ribosomal subunits was also observed. We thus hypothesized, that rpS5 N-terminal truncation likely results in a slow dissociation of eIF2 from 48S complexes, thus also causing accumulation of eIF5B and in turn leading to compromised subunit joining. Therefore, truncation of rpS5 causes mutant ribosomes leaky-scan through the uORF1 AUG, making it further impossible for the scanning subunits to bypass uORFs 2, 3 or 4 and re-initiate at AUG codon 5 to translate the GCN4 ORF.
Keywords: ribosomal protein, GCN4, translation
Abstract:
Long Non-coding RNAs have recently been the focus of many studies due to their apparently ubiquitous regulatory role in almost all higher eukaryotes. In order to gain insight into the features that may help us differentiate this group of regulatory RNAs from the protein-coding ones, we have created a relational database of studied non-coding RNAs which have been proven to have a cellular function. We have analyzed the different features of these lncRNAs and have compared them with a control group of protein-coding messages that were randomly chosen using the BIOMART random picks feature. The analysis logic and visualization layer were implemented in Python language. The selected RNAs were analyzed for the various features of their predicted ORFs, miRNAs and splicing patterns. The outcome of the analysis indicated differences in the splicing patterns, sequence composition of the RNAs and the size and sequence patterns of the predicted ORFs in the two groups. Some of these differences can be used to differentiate the protein-coding messages from the lncRNAs with a high degree of certainty. The results of these analyses will be presented in detail.
Keywords: lncRNA, miRNA, Pattern
Abstract:
The Bacillus subtilis phage phi29 packaging motor requires prohead RNA for genome encapsidation. The NMR structure of the prohead RNA E-loop hairpin, (5’AUUGAGUU), and MC-SYM predictions for prohead RNA E-loops provide a basis for comparative analysis of hairpin structures in prohead and ribosomal RNA. All the hairpins contain a U-turn motif but differ in the first non-canonical pair and backbone orientation. These structures provide benchmarks for further improvements in RNA structure predictions from sequence.
Keywords: prohead RNA, NMR, RNA structure prediction
