RRM
2012 Rustbelt RNA Meeting
RRM
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Talk abstracts
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
Background: The addition of 5’ cap is the first step in pre-mRNA processing and this reaction is catalyzed by the nuclear capping enzyme associated with RNA pol II. The cap plays a central role in subsequent steps of processing steps and in translation initiation, and loss of cap was thought to irreversibly commit mRNA to decay. In contrast to this notion, our lab identified a ~140 kDa cytoplasmic complex of capping enzyme together with a 5’-monophosphate RNA kinase that converts uncapped mono-phosphate RNAs to diphosphate substrates for mRNA capping enzyme. Cytoplasmic capping functions to control the cycling of the target mRNAs between translating and non-translating states, and plays a role in cellular recovery from stress as overexpression of inactive capping enzyme reduces the ability of cells to recover from oxidative stress.
Principal Finding: In the present study we show Nck1, a SH3/SH2 adaptor protein binds directly with cytoplasmic capping enzyme. The association is RNase independent and requires the third SH3 domain of Nck1. The proline rich sequence in both N-terminal and C-terminal domains of capping enzyme are potential sites for Nck1 binding, and we show that the C-terminal domain of capping enzyme (572-597) is required for Nck1 interaction as well as for cytoplasmic capping. The reconstitution of a functional complex following addition of recombinant Nck1 to a cytoplasmic extract prepared from cells expressing epitope tagged capping enzyme provided direct evidence of a role for Nck1 in assembly of the cytoplasmic capping enzyme complex. Finally, reduced activity of immunoprecipitated cytoplasmic capping enzyme complex from cells in which Nck1 was knocked down provided functional evidence of a role for Nck1 in cytoplasmic capping.
Conclusion: Our results identify a previously unknown interaction between Nck1 and cytoplasmic capping enzyme. We suggest Nck1 serves as the scaffold for the recruiting of the 5’-monophosphate RNA kinase to the cytoplasmic capping enzyme. Experiments are in progress to identify the 5’-monophosphate RNA kinase. Our study establishes that Nck1, an adapter protein known to mediate receptor tyrosine kinase signaling at the membrane level, also regulates gene expression through cytoplasmic capping.
Supported by grant R01 GM084177 from the National Institute of General Medical Sciences, National Institutes of Health.
Keywords: Cytoplasmic capping, Cytoplasmic capping enzyme, SH3 domain
Abstract:
Alternative splicing greatly enhances the diversity of proteins encoded by eukaryotic genomes, and is also important in gene expression control. In contrast to the great depth of knowledge as to molecular mechanisms in the splicing pathway itself, relatively little is known about the regulatory events behind this process. The 5’-UTR and 3’-UTR in pre-mRNAs play a variety of roles in controlling eukaryotic gene expression, including translational modulation, and nearly 4,000 of the roughly 14,000 protein coding genes in Drosophila contain introns of unknown functional significance in their 5’-UTR. Here we report the results of an RNA electrophoretic mobility shift analysis of Drosophila rnp-4f 5’-UTR intron 0 splicing regulatory proteins. The pre-mRNA potential regulatory element consists of an evolutionarily- conserved 177-nt stem-loop arising from pairing of intron 0 with part of adjacent exon 2. Incubation of in vitro transcribed probe with embryo protein extract is shown to result in two shifted RNA-protein bands, and protein extract from a dADAR null mutant fly line results in only one shifted band. A mutated stem-loop in which the conserved exon 2 primary sequence is changed but secondary structure maintained by introducing compensatory base changes results in diminished band shifts. To test the hypothesis that dADAR plays a role in intron splicing regulation in vivo, levels of unspliced rnp-4f mRNA in dADAR mutant were compared to wild-type via real-time qRT-PCR. The results show that during embryogenesis unspliced rnp-4f mRNA levels fall by up to 85% in the mutant, in support of the hypothesis. Taken together, these results demonstrate a novel role for dADAR protein in rnp-4f 5’-UTR alternative intron splicing regulation which is summarized in a working model.
Keywords: Alternative splicing regulation, cis- & trans-regulatory elements, dADAR function
Abstract:
Adenosine deaminases that act on RNA (ADARs) are a small family of proteins that modify dsRNAs in the mammalian brain. These enzymes edit adenosine to inosine, interpreted as guanosine by translational machinery, in serotonin and glutamate receptor B (GluR-B) pre-mRNAs in humans. Two editing events in the GluR-B RNA are known to modify ion channel permeability and resensitization kinetics. The enzyme that produces these mutations in humans is ADAR2, however, little is known concerning the high degree of specificity with which ADARs select their substrates. To better understand the elements of substrate selection and structurally predict other substrates we sought to 1) determine a co-crystal structure of ADAR2 with RNA and 2) determine the structure of the unstructured loop of the catalytic domain by making mutants in order to understand its function. We have generated crystals of GluR-B mimics with an ADAR2 truncation known as PP-R2D with the help of an Art Robbins Phoenix LHS; screening to produce larger crystals suitable for x-ray diffraction is ongoing. We have solved two mutant structures, S458G and R455A, of the catalytic domain of ADAR2 to define a stable structure in an area of previously undefined electron density and identify the biochemical function of this unstructured loop of the protein near the site of catalysis. These structures remain incomplete, yet provide a better descriptor of the dynamicity of the loop. To further understand the kinetic role of the loop we have replaced it with several glycines and are comparing its kinetic activity to the wild type enzyme via poison primer assays employing a fluorescent label. The results of these studies have shown that large complexes of ADAR2 and substrate mimics can be crystallized, which is fortuitous for further structure determination, and that the unstructured loop of the catalytic domain remains highly dynamic despite mutations to decrease mobility.
Keywords: Adenosine Deaminase, RNA editing, Glutamate receptor
Abstract not available online - please check the printed booklet.
Abstract:
Androgen receptor (AR) is a transcription factor and a member of the steroid receptor family. AR plays an important role in the regulation of both prostate cell proliferation and growth suppression. AR is a major target for Prostate Cancer treatment. AR mRNA contains a 6.8 kb long 3’ untranslated regions (UTR) and it is a target of many miRNAs. Targeting of AR by miRNAs is likely to modulate differential regulation of AR expression. We have shown that AR is a target of miR-488* and miR-644a. In this study, we are investigating the combinatorial action of miRNAs on AR expression. The repression of AR expression is substantially enhanced in prostate cancer cells treated with both miRNAs in comparison with the individual miRNAs. Similar additive effect was also observed in cancer cell apoptosis and viability. In addition, we are investigating effects of AR 3’ UTR in AR expression using luciferase reporter system. Preliminary data demonstrates that the expression of Luciferase containing longer UTRs is suppressed. In summation, our data indicates the significance of miRNA and UTR in AR gene regulation at posttranscriptional levels.
Keywords: UTR, miRNA, Androgen Receptor
Abstract not available online - please check the printed booklet.
Abstract:
PUFs are unique RNA-binding proteins with incredible specificity and high affinity for their mRNA targets. PUFs limit gene expression through induction of mRNA decay and translational inhibition. PUF proteins are present across eukaryotes, and are involved in a myriad of important biological processes including stem cell maintenance, learning and memory, and embryonic development. I study the founding PUF family member, Pumilio (Pum), which binds to UGUANAUA sequences in the Drosophila transcriptome. Binding to this element in the Hunchback (Hb) mRNA 3 UTR, Pum creates an anterior-posterior gradient of Hb protein which confers correct abdominal segmentation in the developing Drosophila embryo. I have developed an assay which measures Pum repression in a Drosophila cell line. Using this assay, I have shown that Pum employs multiple strategies to block expression of its targets. The Zn finger protein Nanos was originally thought to be required for Pum repression, but I have discovered that Nanos merely serves to robustly activate repression through Pums RNA-binding domain (RBD). By itself, the RBD bears very minimal activity without Nanos, and is weaker than the full length protein even when stimulated. Consequently, I have also identified multiple autonomous repressor domains in the Pum N-terminus that inhibit translation in a manner independent of Nanos and other known corepressors. These regions are auto-regulated by conserved motifs within Pums N-terminus. This work is an important step in understanding how PUFs can fine-tune mRNA expression by utilizing multiple mechanisms that act in concert via repression domains appended to its highly conserved RBD.
Keywords: PUF, translation inhibition, mRNA repression
Abstract not available online - please check the printed booklet.
Abstract:
Albomycin δ2 is a potent antibiotic produced by Streptomyces sp. ATCC 700974. Albomycin enters the bacterial cell using a ferrichrome transporter and releases a seryl-tRNA synthetase inhibitor on cleavage by cellular proteases. This “Trojan horse” mechanism allows it to stall bacterial protein synthesis at very low MICs, <~0.005 µg/mL in case of E.coli. Albomycin possesses a peptidyl nucleoside structure with unique modifications of N4-carbamylation and N3-methylation on cytosine. Cytosine is linked to a thioxylo-furanose ring which has not been reported in any other natural/synthetic product. The albomycin (abm) biosynthetic gene cluster was identified from Streptomyces sp. ATCC 700974 using a probe based on sidA, encoding an N5-L-Ornithine monooxygenase, a gene involved in ferrichrome biosynthesis. Genes encoding a methyltransferase (abmI) and a carbamoyltransferase (abmE) were found in the gene cluster. A homologous gene cluster (ctj) was found in the sequenced genome of Streptomyces sp.C . The ctj gene cluster also encodes methyltransferase(ctjF) and carbamoyltransferase (ctjE) homologs. The ctj gene cluster likely encodes a Trojan horse antibiotic with a modified nucleobase and thioxylo-furanose ring. A ΔabmE mutant of Streptomyces sp. ATCC 700974 strain resulted in a loss of production of wild type albomycin δ2. Heterologously expressed AbmE was shown to carbamylate the cytosine nucleoside of an unmodified albomycin intermediate at the N4 position in vitro. Wild type albomycin production in the ΔabmE mutant was restored by ctjE or abmE on plasmid. A ΔabmI mutant strain produced a desmethyl-descarbamoyl analogue of albomycin. AbmI, heterologously expressed and purified from E.coli, flexible in substrate specificity, was shown to methylate a variety of cytosine nucleosides. In summary, we discovered and identified the gene cluster involved in albomycin biosynthesis . The discovery of two nucleoside base modification genes involved in albomycin biosynthesis sets the stage to apply them to modify nucleoside antibiotics.
References:
1.Zeng Y, Kulkarni A,Yang Z,Patil PB, Zhou W, Chi X,Van Lanen S,Chen S. Biosynthesis of Albomycinδ2 Provides a Template for Assembling Siderophore and Aminoacyl-tRNA Synthetase Inhibitor Conjugates. ACS Chemical Biology DOI: 10.1021/cb300173x
2. Yu Zeng, Hervé Roy, Preeti B. Patil, Michael Ibba, and Shawn Chen Characterization of Two Seryl-tRNA Synthetases in Albomycin-Producing Streptomyces sp. Strain ATCC 700974 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Nov. 2009, p. 4619–4627
Keywords: Nucleoside antibiotics, Base Modification, tRNA synthetase inhibitor
Abstract:
Personalized medicine is expected to benefit from the combination of genomic information with the global monitoring of molecular components and physiological states. To ascertain whether this can be achieved, we determined the whole genome sequence of an individual at high accuracy and performed an integrated Personal Omics Profiling (iPOP) analysis, combining genomic, transcriptomic, proteomic, metabolomic, and autoantibodyomic information, over a 21-month period that included healthy and two virally infected states. Our iPOP analysis of blood components revealed extensive, dynamic and broad changes in diverse molecular components and biological pathways across healthy and disease conditions. Importantly, genomic information was also used to estimate medical risks, including Type 2 Diabetes, whose onset was observed during the course of our study. Our study demonstrates that longitudinal personal omics profiling can relate genomic information to global functional omics activity for physiological and medical interpretation of healthy and disease states.
Keywords: Genomics
Abstract:
Helix 69 (H69) is a 19-nucleotide stem-loop segment in bacterial 23S ribosomal RNAs (rRNAs). The sequence, secondary structure, and the post-transcriptional modifications are highly conserved. Being part of a key intersubunit bridge, B2a, and interacting with multiple translational factors during protein synthesis, H69 has become an attractive target for new antibiotics. It was shown through biophysical and biochemical studies that pseudouridine modifications confer effects on both the structure and function of H69; however, the structural effects of pseudouridylation are difficult to assess without a structure of the unmodified H69. In this project, the structures of unmodified and pseudouridylated H69s in aqueous solution were solved by nuclear magnetic resonance spectroscopy. Comparison of the two structures suggests that profound conformational changes are induced by pseudouridylations, and modifications at the three sites (1911, 1915, and 1917, E. coli numbering) work synergistically to modulate H69 folding. Results from this research not only provide insight into the structural effects of pseudouridylations on RNA folding, but also establish a platform to explore the binding patterns between the H69s, unmodified and pseudouridylated, and potential antibiotic candidates.
Keywords: RNA, pseudouridine, structure
Abstract:
Human Enterovirus 71 (EV71), the causative agent of hand, foot, and mouth disease, has been shown to recruit the small ribosomal unit through a type I IRES. Recent experiments have shown hnRNP A1 (A1) trans activates the EV71 translation by binding to stem loops II and VI. In an effort to better understand how A1 stimulates EV71 translation, we sought to characterize protein-SLII interactions using a combination of biophysical and functional biochemical assays.
The high affinity binding site for A1 is UAGGGU, with UAG being the primary binding element. Examination of the predicted secondary structure of SLII showed that a UAG element is located within a 5 nt bulge of the stem loop, which we hypothesized to be the specific A1 binding site.
A construct of EV71 SLII was in vitro transcribed and analyzed by 2D NMR spectroscopy. The 2D 1H-1H NOE patterns clearly show that the isolated SLII construct folds as predicted. Isothermal titration calorimetry (ITC) experiments were done with a truncated version of A1 (UP1), which contains the two RRM domains connected by a flexible linker. While it was initially assumed SLII had just one binding site for UP1, the experiments showed binding at two sites. Binding at both sites was found to be extremely tight, with binding at the first sight taking place in the picomolar range, and binding at the second sight taking place in the nanomolar range.
Since UP1 primarily recognizes single stranded RNA, it was hypothesized that the hairpin loop contains the second binding site. Mutations were introduced in both the bulge and hairpin loop. ITC experiments confirmed the involvement of both elements in UP1 binding, while NMR experiments showed that the mutations did not affect the RNA secondary structure. Furthermore, the significance of the bulge loop to IRES activity was probed using an in vivo translation assay. Substitution of the bulge UAG sequence with a CCC sequence reduced IRES activity by more than 80% relative to wild type levels.
References:
Shih, S.R., Stollar, V., and Li, M.L. (2011) Host Factors in EV71 Replication. J. Virol. 85(19):9658-9666.
Lin, J.Y., Shih, S.R., Pan, M., Li, C., Lue, C.F., Stollar, V., and Li, M.L. (2009) hnRNP A1 interacts with the 5’ Untranslated Regions of Enterovirus 71 and Sindbis Virus RNA and Is Required for Viral Replication. J. Virol. 83(12):6106-6114.
Keywords: IRES, hnRNP A1
Abstract not available online - please check the printed booklet.
Abstract:
The Dimerization Initiation Sequence (DIS) is a conserved hairpin-loop motif on the 5’ UTR of the HIV-1 genome. It plays an important role in genome dimerization through formation of a “kissing complex” intermediate between two homologous DIS sequences. This bimolecular kissing complex ultimately leads to the formation of an extended RNA duplex. Understanding the kinetics of this interaction is key to exploiting DIS as a possible drug target against HIV.
Here, we report a novel study that makes an important contribution to understanding the dimerization mechanism of HIV-1 RNA in vitro. Our work has employed single-molecule fluorescence resonance energy transfer to monitor the dimerization of minimal HIV-1 RNA sequence containing DIS. Most significantly, we observe a previously uncharacterized folding intermediate that plays a critical role in the dimerization mechanism. Our data clearly show that dimerization involves three distinct steps in dynamic equilibrium and regulated by Mg2+ ions. Two of the steps correspond to previously proposed structures: the kissing complex and the extended duplex. Surprisingly, our data reveal a previously unobserved obligatory folding intermediate, consistent with a bent kissing complex conformation, similar to the TAR-TAT complex. Mutations of the highly conserved purines flanking the DIS loop destabilize this intermediate, indicating that these purines may play an important role in the HIV-1 RNA dimerization in vivo.
Keywords: FRET, HIV-1 RNA Dimerization, kissing complex
Abstract not available online - please check the printed booklet.
Abstract:
RNA 3D Hub (http://rna.bgsu.edu/rna3dhub) is a new online resource designed to make the RNA 3D structural data deposited in the PDB/NDB more accessible and useful to RNA scientists.
The PDB serves as the international repository of atomic-resolution 3D structures and contains a separate entry for each solved structured deposited by researchers that meets the data quality requirements. Many of these structures are redundant as they represent the same molecule from the same organism.
To address this problem, RNA 3D Hub hosts non-redundant sets of RNA-containing 3D structures and structural annotations of crucial pairwise interactions found in 3D structures, including base-pairing, -stacking, and -backbone interactions. These non-redundant lists of RNA 3D structures are produced by an automated procedure based on sequence similarity, 3D superpositions, and structure quality considerations.
RNA 3D Hub also contains RNA 3D motifs extracted from all atomic-resolution RNA 3D structures deposited in PDB, and the RNA 3D Motif Atlas, a representative collection of RNA 3D motifs.
RNA 3D motifs are recurrent structural modules that are essential for many biological functions and RNA folding. Usually drawn as unstructured hairpin and internal “loops”, these motifs are organized by non-canonical basepairs, supplemented by characteristic stacking and base-backbone interactions.
To create the Motif Atlas we extract RNA 3D motif instances from the current non-redundant list using FR3D, a program for symbolic and geometric searching of RNA 3D structures. Next, we use a clustering approach to obtain a representative collection of RNA 3D motifs. All these data are stored in RNA 3D Hub and can be accessed online. Unique and stable ids are assigned to all motifs, to all motif instances, and to all non-redundant equivalence classes of structure files.
RNA 3D Hub is updated automatically on a regular schedule, and a versioning system is implemented to provide independent access to data snapshots. RNA 3D Hub also provides a rich user interface to allow for efficient data visualization.
Keywords: RNA 3D structure, online database, RNA 3D motifs
Abstract:
We discovered a phi29 pRNA three-way junction (3WJ) motif with unusual thermodynamic stability. The ΔG of the nanoparticles is extremely low and the slope of the melting temperature curve of the three-fragment RNA complex is close to 90°. The motif was used as a RNA scaffold to construct bi-, tri-, and tetra-valent RNA nanoparticles with very high chemical and thermodynamic stability (1-3). The resulting RNA nanoparticles are resistant to denaturation in 8M urea and, do not dissociate at ultra-low concentrations in vitro and in vivo. Each arm of the three-way junction (3WJ) or X-motif can harbor one siRNA, ribozyme, miRNA or aptamer without affecting the folding of the central core, and each daughter RNA molecule within the nanoparticle fold into respective authentic structure, and retain their independent function (Fig. 1). Epigenetic regulation effects progressively increased with increasing number of functional modules in the nanoparticle. More importantly, systemic injection of the ligand-containing nanoparticles into the tail-vein of mice revealed that the RNA nanoparticles remained intact without showing any sign of dissociation or degradation; and strongly bound to cancers without detectable entry into liver, lung or other organs or tissues. Pharmacokinetic analysis in mice revealed that the half-life of RNA nanoparticles was extended 10-fold compared to the siRNA counterpart, and did not induce cytokine, interferon, antibody, and toxicity while retaining favorable biodistribution and pharmacokinetics profiles.
References:
1. 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; 6:658.
2. 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.
3. Guo P. The emerging field of RNA nanotechnology. Nature Nanotech. 2010. 5:833.
Keywords: RNA nanoparticle, three-way junction, X-motif
Abstract:
In all three domains of life, the RNA subunit of the ribonucleoprotein form of RNase P is the catalyst responsible for Mg2+-dependent endonucleolytic 5′ processing of precursor tRNAs (pre-tRNAs). In silico searches have identified eukaryotic genes that encode the RNase P RNA (RPR). While several eukaryotic RPRs studied thus far (human, yeast and zebrafish) are pol III transcripts, the Drosophila RPR is not detected in a pol III-specific chromatin immuno-precipitation (ChIP) assay. Interestingly, the annotated RPR gene is present in the second intron of the uncharacterized protein-coding gene CG1746. The overall RPR-coding region is highly conserved in all 12 Drosophila species, however, the 5′ and 3′ flanking regions are poorly conserved and lack signature elements of a pol III transcript such as the TATA box and a poly-T terminator. The intronic RPR (iRPR) is expresed throughout development. In biochemical assays, we found that iRPR co-purifies with the active native RNase P holoenzyme from Drosophila cells. Not only did the iRPR alone show activity in vitro, an RNA oligonucleotide complementary to its sequence effectively inhibited the pre-tRNA processing activity of the partially-purified RNase P holoenzyme, confirming that it is the catalytic moiety. To investigate how this iRPR is processed from the spliced intron of CG1746, we have developed a red fluorescent protein (RFP)-based reporter assay. By introducing the intron encoding the iRPR into the RFP-coding region, we have been able to use RFP splicing and expression as a direct monitor of iRPR biogenesis in Drosophila cells. These studies are expected to provide insights into the possible coupling of iRPR biogenesis to pol II transcription and mRNA splicing, and also highlight the regulatory web around this ancient ribozyme.
Keywords: RNase P, Drosophila, Splicing
Abstract:
Pseudouridine Synthases (ΨS) post-transcriptionally modify uridine (U) to its C-glycosidic isomer pseudouridine (Ψ) and are divided into six families based on sequence and structural similarity.1 Conserved active site residues in all the ΨS families indicate the likelihood of a common mechanism across the families. Mechanistic insight has come from studies of the action of ΨS on RNA containing 5-fluorouridine ([F5U]RNA).2, 3 RluA and TruA are inhibited by [F5U]RNA forming a gel shifted band, but TruB treats [F5U]RNA as a substrate and does not form an adduct.4 Analysis of the target F5U after the action of ΨS indicated a hydrated product, and 18O labeling studies indicated that the F5U product was hydrated directly by water from solution and rather than from hydrolysis of an ester linkage as had been proposed. Furthermore, work with TruA indicated that the label was incorporated upon heat disruption, suggesting that the adduct was covalent but collapsed upon heating to an F5U product species that subsequently underwent spontaneous hydration.5
The current report extends the 18O wash-in and gel shift studies to the interaction between RluA and [F5U]RNA. RluA forms an adduct with [F5U]ASL (RluA-[F5U]ASL) as judged by its behavior on SDS- and urea-PAGE gels.4 Intriguingly, when the isolated product RNA containing the rearranged and hydrated F5U is incubated with RluA, an adduct forms with gel behavior identical to the original RluA-[F5U]ASL adduct. Moreover, 18O wash-in studies indicate that the label is incorporated during adduct formation, not heat disruption. These observations point toward a non-covalent nature for the adduct. However, when the essential active site aspartic acid is replaced with asparagine, the adduct is no longer formed either with the substrate or product [F5U]ASL. These observations will be presented and discussed in the context of the mechanism of pseudouridine synthases.
References:
1. Mueller, E. G.; Ferre-DAmare, A. R., Pseudouridine Formation, the Most Common Transglycosylation in RNA. Landes Bioscience: Austin,Tx, 2009.
2. Miracco, E. J.; Mueller, E. G., The Products of 5-Fluorouridine by the Action of the Pseudouridine Synthase TruB Disfavor One Mechanism and Suggest Another. J Am Chem Soc 2011, 133, (31), 11826-9.
3. Spedaliere, C. J.; Ginter, J. M.; Johnston, M. V.; Mueller, E. G., The Pseudouridine Synthases: Revisiting a Mechanism That Seemed Settled. J Am Chem Soc 2004, 126, (40), 12758-9.
4. Spedaliere, C. J.; Mueller, E. G., Not All Pseudouridine Synthases are Potently Inhibited by RNA Containing 5-Fluorouridine. RNA 2004, 10, (2), 192-9.
5. McDonald, M. K.; Miracco, E. J.; Chen, J.; Xie, Y.; Mueller, E. G., The Handling of the Mechanistic Probe 5-Fluorouridine by the Pseudouridine Synthase TruA and Its Consistency with the Handling of the Same Probe by the Pseudouridine Synthases TruB and RluA. Biochemistry 2010, 426-36.
Keywords: Pseudouridine Synthase, RNA Modifications, Enzymology
Abstract:
The global identification of individual transfer ribonucleic acids (tRNAs) can prove to be very difficult, requiring rigorous sample preparation prior to analysis. Here, we illustrate a highly optimized MS/MS assay for rapid global identification of tRNAs, building on techniques previously introduced by our group. The goal is to identify sequences of oligonucleotides that are unique to a certain isoacceptor tRNA by detecting unique fragmentation channels. This approach enables accurate tRNA identification in a manner more conducive to high-throughput analyses. Our results show 43 out of the 47 isoaccepting tRNAs predicted to be present in E. coli are routinely detected by targeting 22 precursor ions in less than 15 minutes. Validation experiments included the examination of tRNAs from a tRNA over-expression system. The approach is general enough that it could be applied to archaeal and eukaryotic organisms, and this approach should also be feasible for the targeted analysis of other small RNAs in the cell.
Keywords: tRNA, Reverse phase chromatography, Tandemn Mass Spectrometry
Abstract not available online - please check the printed booklet.
Abstract:
Despite their high tRNA selectivity, many aminoacyl-tRNA synthetases mis-activate non-cognate amino acids that are structurally similar to their cognate substrate. For example, prolyl-tRNA synthetase (ProRS) fails to effectively discriminate between cognate Pro and non-cognate Ala and Cys. In most bacteria, mischarged Ala-tRNAPro is hydrolyzed in a catalytic domain distinct from the synthetic active site (INS). In contrast, Cys-tRNAPro, is cleared by YbaK, a single domain homolog of INS. Although most bacteria employ this “triple-sieve” editing mechanism, some bacterial species encode a ProRS that lacks the INS domain. Five distinct families of INS homologs collectively known as the YbaK superfamily have now been identified (YbaK, PrdX, ProX, PA2301, and YeaK), and alternative triple-sieve editing mechanisms exist. For example, Caulobacter crescentus (Cc) lacks a full-length INS domain and relies on the trans editing activities of both YbaK (Cys editing) and PrdX (Ala editing). In addition, YbaK, PrdX, and INS use divergent mechanisms to recognize their aa-tRNAPro substrates. PrdX recognizes the discriminator base (A73) and the first (C1:G72) base pair of tRNAPro, and also collaborates with elongation factor Tu to ensure hydrolysis of only Ala-tRNAPro and not Ala-tRNAAla. In contrast, INS and YbaK do not appear to recognize specific acceptor stem elements. Whereas the INS domain of ProRS depends on correct anticodon recognition to hydrolyze only Ala-tRNAPro, YbaK interacts with ProRS to ensure specific cleavage of Cys-tRNAPro and not Cys-tRNACys. Taken together, these data highlight the diversity of mechanisms used by the YbaK superfamily to maintain the fidelity of Pro codon translation.
Keywords: tRNA, aminoacyl-tRNA synthetases, editing
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
