RNA

Escherichia coli tmRNA lacking pseudoknot 1 tags truncated proteins in vivo and in vitro.

Publication Date: 2008 Nov 10 PMID: 19001120
Authors: Wower, I. K. - Zwieb, C. - Wower, J.
Journal: RNA

Transfer-messenger RNA (tmRNA) and protein SmpB facilitate trans-translation, a quality-control process that tags truncated proteins with short peptides recognized by a number of proteases and recycles ribosomes stalled at the 3' end of mRNA templates lacking stop codons. The tmRNA molecule is a hybrid of tRNA- and mRNA-like domains that are usually connected by four pseudoknots (pk1-pk4). Replacement of pk1 with a single-stranded RNA yields pk1L, a mutant tmRNA that tags truncated proteins very poorly in vitro but very efficiently in vivo. However, deletion of the whole pk1 is deleterious for protein tagging. In contrast, deletion of helix 4 yields Deltah4, a fully functional tmRNA derivative containing a single hairpin instead of pk1. Further deletions in the pk1 segment yield two subclasses of mutant tmRNAs that are unable to tag truncated proteins, but some of them bind to stalled ribosomes. Our studies demonstrate that pk1 is not essential for tmRNA functions but contributes to the stability of the tmRNA structure. Our studies also indicate that the length of this RNA segment is critical for both tmRNA binding to the ribosome and resumption of translation.

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Molecular characterization of human Argonaute-containing ribonucleoprotein complexes and their bound target mRNAs.

Publication Date: 2008 Oct 31 PMID: 18978028
Authors: Landthaler, M. - Gaidatzis, D. - Rothballer, A. - Chen, P. Y. - Soll, S. J. - Dinic, L. - Ojo, T. - Hafner, M. - Zavolan, M. - Tuschl, T.
Journal: RNA

microRNAs (miRNAs) regulate the expression of mRNAs in animals and plants through miRNA-containing ribonucleoprotein particles (RNPs). At the core of these miRNA silencing effector complexes are the Argonaute (AGO) proteins that bind miRNAs and mediate target mRNA recognition. We generated HEK293 cell lines stably expressing epitope-tagged human AGO proteins and other RNA silencing-related proteins and used these cells to purify miRNA-containing RNPs. Mass spectrometric analyses of the proteins associated with different AGO proteins revealed a common set of helicases and mRNA-binding proteins, among them the three trinucleotide repeat containing proteins 6 (TNRC6A,-B,-C). mRNA microarray analyses of these miRNA-associated RNPs revealed that AGO and TNRC6 proteins bind highly similar sets of transcripts enriched in binding sites for highly expressed endogenous miRNAs, indicating that the TNRC6 proteins are a component of the mRNA-targeting miRNA silencing complex. Together with the very similar proteomic composition of each AGO complex, this result suggests substantial functional redundancy within families of human AGO and TNRC6 proteins. Our results further demonstrate that we have developed an effective biochemical approach to identify physiologically relevant human miRNA targets.

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SMG6 is the catalytic endonuclease that cleaves mRNAs containing nonsense codons in metazoan.

Publication Date: 2008 Oct 30 PMID: 18974281
Authors: Huntzinger, E. - Kashima, I. - Fauser, M. - Sauliere, J. - Izaurralde, E.
Journal: RNA

Messenger RNAs harboring nonsense codons (or premature translation termination codons [PTCs]) are degraded by a conserved quality-control mechanism known as nonsense-mediated mRNA decay (NMD), which prevents the accumulation of truncated and potentially harmful proteins. In Drosophila melanogaster, degradation of PTC-containing messages is initiated by endonucleolytic cleavage in the vicinity of the nonsense codon. The endonuclease responsible for this cleavage has not been identified. Here, we show that SMG6 is the long sought NMD endonuclease. First, cells expressing an SMG6 protein mutated at catalytic residues fail to degrade PTC-containing messages. Moreover, the SMG6-PIN domain can be replaced with the active PIN domain of an unrelated protein, indicating that its sole function is to provide endonuclease activity for NMD. Unexpectedly, we found that the catalytic activity of SMG6 contributes to the degradation of PTC-containing mRNAs in human cells. Thus, SMG6 is a conserved endonuclease that degrades mRNAs terminating translation prematurely in metazoa.

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Evidence of a novel RNA secondary structure in the coding region of HIV-1 pol gene.

Publication Date: 2008 Oct 31 PMID: 18974280
Authors: Wang, Q. - Barr, I. - Guo, F. - Lee, C.
Journal: RNA

RNA secondary structures play several important roles in the human immunodeficiency virus (HIV) life cycle. To assess whether RNA secondary structure might affect the function of the HIV protease and reverse transcriptase genes, which are the main targets of anti-HIV drugs, we applied a series of different computational approaches to detect RNA secondary structures, including thermodynamic RNA folding predictions, synonymous variability analysis, and covariance analysis. Each method independently revealed strong evidence of a novel RNA secondary structure at the junction of the protease and reverse transcriptase genes, consisting of a 107-nucleotide region containing three stems, A, B, and C. First, RNA folding calculations by mfold and RNAfold both predicted the secondary structure with high confidence. Moreover, the same structure was predicted in a diverse set of reference sequences in HIV-1 group M, indicating that it is conserved across this group. Second, the predicted base-pairing regions displayed markedly reduced synonymous variation (approximately threefold lower than average) in a data set of 20,000 HIV-1 subtype B sequences from clinical samples. Third, independent analysis of covariation between synonymous mutations in this data set identified 10 covariant mutation pairs forming two diagonals that corresponded exactly to the sites predicted to base-pair in stems A and B. Finally, this structure was validated experimentally using selective 2'-hydroxyl acylation and primer extension (SHAPE). Discovery of this novel secondary structure suggests many directions for further functional investigation.

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The secondary structure of the 5' end of the FIV genome reveals a long-range interaction between R/U5 and gag sequences, and a large, stable stem-loop.

Publication Date: 2008 Oct 30 PMID: 18974279
Authors: Kenyon, J. C. - Ghazawi, A. - Cheung, W. K. - Phillip, P. S. - Rizvi, T. A. - Lever, A. M.
Journal: RNA

Feline immunodeficiency virus (FIV) is a lentivirus that infects cats and is related to human immunodeficiency virus (HIV). Although it is a common worldwide infection, and has potential uses as a human gene therapy vector and as a nonprimate model for HIV infection, little detail is known of the viral life cycle. Previous experiments have shown that its packaging signal includes two or more regions within the first 511 nucleotides of the genomic RNA. We have undertaken a secondary structural analysis of this RNA by minimal free-energy structural prediction, biochemical mapping, and phylogenetic analysis, and show that it contains five conserved stem-loops and a conserved long-range interaction between heptanucleotide sequences 5'-CCCUGUC-3' in R/U5 and 5'-GACAGGG-3' in gag. This long-range interaction is similar to that seen in primate lentiviruses where it is thought to be functionally important. Along with strains that infect domestic cats, this heptanucleotide interaction can also occur in species-specific FIV strains that infect pumas, lions, and Pallas' cats where the heptanucleotide sequences involved vary. We have analyzed spliced and genomic FIV RNAs and see little structural change or sequence conservation within single-stranded regions of the 5' UTR that are important for viral packaging, suggesting that FIV may employ a cotranslational packaging mechanism.

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Genome 3'-end repair in dengue virus type 2.

Publication Date: 2008 Oct 30 PMID: 18974278
Authors: Teramoto, T. - Kohno, Y. - Mattoo, P. - Markoff, L. - Falgout, B. - Padmanabhan, R.
Journal: RNA

Genomes of RNA viruses encounter a continual threat from host cellular ribonucleases. Therefore, viruses have evolved mechanisms to protect the integrity of their genomes. To study the mechanism of 3'-end repair in dengue virus-2 in mammalian cells, a series of 3'-end deletions in the genome were evaluated for virus replication by detection of viral antigen NS1 and by sequence analysis. Limited deletions did not cause any delay in the detection of NS1 within 5 d. However, deletions of 7-10 nucleotides caused a delay of 9 d in the detection of NS1. Sequence analysis of RNAs from recovered viruses showed that at early times, virus progenies evolved through RNA molecules of heterogeneous lengths and nucleotide sequences at the 3' end, suggesting a possible role for terminal nucleotidyl transferase activity of the viral polymerase (NS5). However, this diversity gradually diminished and consensus sequences emerged. Template activities of 3'-end mutants in the synthesis of negative-strand RNA in vitro by purified NS5 correlate well with the abilities of mutant RNAs to repair and produce virus progenies. Using the Mfold program for RNA structure prediction, we show that if the 3' stem-loop (3' SL) structure was abrogated by mutations, viruses eventually restored the 3' SL structure. Taken together, these results favor a two-step repair process: non-template-based nucleotide addition followed by evolutionary selection of 3'-end sequences based on the best-fit RNA structure that can support viral replication.

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The role of the putative 3' end processing endonuclease Ysh1p in mRNA and snoRNA synthesis.

Publication Date: 2008 Oct 29 PMID: 18971324
Authors: Garas, M. - Dichtl, B. - Keller, W.
Journal: RNA

Pre-mRNA 3' end formation is tightly linked to upstream and downstream events of eukaryotic mRNA synthesis. The two-step reaction involves endonucleolytic cleavage of the primary transcript followed by poly(A) addition to the upstream cleavage product. To further characterize the putative 3' end processing endonuclease Ysh1p/Brr5p, we isolated and analyzed a number of new temperature- and cold-sensitive mutant alleles. We show that Ysh1p plays a crucial role in 3' end formation and in RNA polymerase II (RNAP II) transcription termination on mRNA genes. In addition, we observed a range of additional functional deficiencies in ysh1 mutant strains, which were partially allele-specific. Interestingly, snoRNA 3' end formation and RNAP II termination were defective on specific snoRNAs in the cold-sensitive ysh1-12 strain. Moreover, we observed the accumulation of several mRNAs including the NRD1 transcript in this mutant. We provide evidence that NRD1 autoregulation is associated with endonucleolytic cleavage and that this process may involve Ysh1p. In addition, the ysh1-12 strain displayed defects in RNA splicing indicating that a functional link may exist between intron removal and 3' end formation in yeast. These observations suggest that Ysh1p has multiple roles in RNA synthesis and processing.

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Structure of yeast U6 snRNPs: Arrangement of Prp24p and the LSm complex as revealed by electron microscopy.

Publication Date: 2008 Oct 29 PMID: 18971323
Authors: Karaduman, R. - Dube, P. - Stark, H. - Fabrizio, P. - Kastner, B. - Luhrmann, R.
Journal: RNA

Protein components of the U6 snRNP (Prp24p and LSm2-8) are thought to act cooperatively in facilitating the annealing of U6 and U4 snRNAs during U4/U6 di-snRNP formation. To learn more about the spatial arrangement of these proteins in S. cerevisiae U6 snRNPs, we investigated the structure of this particle by electron microscopy. U6 snRNPs, purified by affinity chromatography and gradient centrifugation, and then immediately adsorbed to the carbon film support, revealed an open form in which the Prp24 protein and the ring formed by the LSm proteins were visible as two separate morphological domains, while particles stabilized by chemical cross-linking in solution under mild conditions before binding to the carbon film exhibited a compact form, with the two domains in close proximity to one another. In the open form, individual LSm proteins were located by a novel approach employing C-terminal genetic tagging of the LSm proteins with yECitrine. These studies show the Prp24 protein at defined distances from each subunit of the LSm ring, which in turn suggests that the LSm ring is positioned in a consistent manner on the U6 RNA. Furthermore, in agreement with the EM observations, UV cross-linking revealed U6 RNA in contact with the LSm2 protein at the interface between Prp24p and the LSm ring. Further, LSmp-Prp24p interactions may be restricted to the closed form, which appears to represent the solution structure of the U6 snRNP particle.

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Crystal structure of an RNA aptamer bound to thrombin.

Publication Date: 2008 Oct 29 PMID: 18971322
Authors: Long, S. B. - Long, M. B. - White, R. R. - Sullenger, B. A.
Journal: RNA

Aptamers, an emerging class of therapeutics, are DNA or RNA molecules that are selected to bind molecular targets that range from small organic compounds to large proteins. All of the determined structures of aptamers in complex with small molecule targets show that aptamers cage such ligands. In structures of aptamers in complex with proteins that naturally bind nucleic acid, the aptamers occupy the nucleic acid binding site and often mimic the natural interactions. Here we present a crystal structure of an RNA aptamer bound to human thrombin, a protein that does not naturally bind nucleic acid, at 1.9 A resolution. The aptamer, which adheres to thrombin at the binding site for heparin, presents an extended molecular surface that is complementary to the protein. Protein recognition involves the stacking of single-stranded adenine bases at the core of the tertiary fold with arginine side chains. These results exemplify how RNA aptamers can fold into intricate conformations that allow them to interact closely with extended surfaces on non-RNA binding proteins.

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Prokaryotic silencing (psi)RNAs in Pyrococcus furiosus.

Publication Date: 2008 Oct 29 PMID: 18971321
Authors: Hale, C. - Kleppe, K. - Terns, R. M. - Terns, M. P.
Journal: RNA

In many prokaryotes, noncoding RNAs that arise from the clustered regularly interspaced short palindromic repeat (CRISPR) loci are now thought to mediate defense against viruses and other molecular invaders by an RNAi-like pathway. CRISPR loci contain multiple short regions of similarity to invader sequences separated by short repeat sequences, and are associated with resistance to infection by corresponding viruses. It is hypothesized that RNAs derived from these regions, termed prokaryotic silencing (psi)RNAs, guide Slicer-like complexes of partner proteins to destroy invader nucleic acids. Here we have investigated CRISPR-derived RNAs in the archaeon Pyrococcus furiosus. Northern analysis revealed multiple RNA species consistent with a proposed biogenesis pathway that includes full-length CRISPR locus transcripts and intermediates generated by endonucleolytic cleavages within the repeat sequences. However, our results identify the principal products of the CRISPR loci as small psiRNAs comprised primarily of invader-targeting sequence with perhaps only 5-10 nucleotides of CRISPR repeat sequence. These RNAs are the most abundant CRISPR RNA species in P. furiosus and are likely the guides for the effector complexes of the proposed prokaryotic RNAi (pRNAi) system. We analyzed cell-free extracts fractionated under non-denaturing conditions and found that the various CRISPR RNA species are components of distinct RNA-protein complexes, including at least two complexes that contain mature-length psiRNAs. Finally, RNAs are produced from all seven CRISPR loci present in the P. furiosus genome, and interestingly, the most recently acquired psiRNAs encoded proximal to the leader sequence of a CRISPR locus appear to be the most abundant.

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Automated motif extraction and classification in RNA tertiary structures.

Publication Date: 2008 Oct 28 PMID: 18957493
Authors: Djelloul, M. - Denise, A.
Journal: RNA

We used a novel graph-based approach to extract RNA tertiary motifs. We cataloged them all and clustered them using an innovative graph similarity measure. We applied our method to three widely studied structures: Haloarcula marismortui 50S (H.m 50S), Escherichia coli 50S (E. coli 50S), and Thermus thermophilus 16S (T.th 16S) RNAs. We identified 10 known motifs without any prior knowledge of their shapes or positions. We additionally identified four putative new motifs.

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Annotation of tertiary interactions in RNA structures reveals variations and correlations.

Publication Date: 2008 Oct 28 PMID: 18957492
Authors: Xin, Y. - Laing, C. - Leontis, N. B. - Schlick, T.
Journal: RNA

RNA tertiary motifs play an important role in RNA folding and biochemical functions. To help interpret the complex organization of RNA tertiary interactions, we comprehensively analyze a data set of 54 high-resolution RNA crystal structures for motif occurrence and correlations. Specifically, we search seven recognized categories of RNA tertiary motifs (coaxial helix, A-minor, ribose zipper, pseudoknot, kissing hairpin, tRNA D-loop/T-loop, and tetraloop-tetraloop receptor) by various computer programs. For the nonredundant RNA data set, we find 613 RNA tertiary interactions, most of which occur in the 16S and 23S rRNAs. An analysis of these motifs reveals the diversity and variety of A-minor motif interactions and the various possible loop-loop receptor interactions that expand upon the tetraloop-tetraloop receptor. Correlations between motifs, such as pseudoknot or coaxial helix with A-minor, reveal higher-order patterns. These findings may ultimately help define tertiary structure restraints for RNA tertiary structure prediction. A complete annotation of the RNA diagrams for our data set is available at http://www.biomath.nyu.edu/motifs/.

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The {beta}-catenin/TCF4 pathway modifies alternative splicing through modulation of SRp20 expression.

Publication Date: 2008 Oct 24 PMID: 18952824
Authors: Goncalves, V. - Matos, P. - Jordan, P.
Journal: RNA

Gene expression programs can become activated in response to extracellular signals. One evolutionarily conserved example is binding of Wnt glycoproteins to their receptor, which triggers a signal transduction cascade that stabilizes cytoplasmic beta-catenin protein, allowing it to translocate into the nucleus. There, beta-catenin binds to TCF/Lef family transcription factors and promotes the expression of target genes. Mutations in either the beta-catenin gene itself or its partner protein APC are responsible for the oncogenic activation of this pathway in colorectal tumors. Here we report the splicing factor SRp20 as a novel target gene of beta-catenin/TCF4 signaling. Transfection of activated beta-catenin mutants into colorectal cells increased expression of endogenous SRp20 transcript and protein and also stimulated a luciferase reporter construct containing the SRp20 gene promoter. In contrast, inhibition of endogenous beta-catenin signaling by a dominant-negative TCF4 construct down-regulated both luciferase reporter and SRp20 expression. We further demonstrate that the beta-catenin/TCF4-mediated increase in SRp20 protein levels is sufficient to modulate alternative splicing decisions in the cells. In particular, we observed a change in the alternative splicing pattern in a control minigene reporter as well as in the endogenous SRp20-regulated CD44 cell adhesion protein. These results demonstrate that the beta-catenin/TCF4 pathway not only stimulates gene transcription, but also promotes the generation of transcript variants through alternative splicing. Our data support the recent notion that transcription and alternative splicing represent two different layers of gene expression and that signaling pathways act upon a coordinated network of transcripts in each layer.

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Archaeal Pus10 proteins can produce both pseudouridine 54 and 55 in tRNA.

Publication Date: 2008 Oct 24 PMID: 18952823
Authors: Gurha, P. - Gupta, R.
Journal: RNA

Pus10, a recently identified pseudouridine (Psi) synthase, does not belong to any of the five commonly identified families of Psi synthases. Pyrococcus furiosus Pus10 has been shown to produce Psi55 in tRNAs. However, in vitro studies have identified another mechanism for tRNA Psi55 production in Archaea, which uses Cbf5 and other core proteins of the H/ACA ribonucleoprotein complex, in a guide RNA-independent manner. Pus10 homologs have been observed in nearly all sequenced archaeal genomes and in some higher eukaryotes, but not in yeast and bacteria. This coincides with the presence of Psi54 in the tRNAs of Archaea and higher eukaryotes and its absence in yeast and bacteria. No tRNA Psi54 synthase has been reported so far. Here, using recombinant Methanocaldococcus jannaschii and P. furiosus Pus10, we show that these proteins can function as synthase for both tRNA Psi54 and Psi55. The two modifications seem to occur independently. Salt concentration dependent variations in these activities of both proteins are observed. The Psi54 synthase activity of M. jannaschii protein is robust, while the same activity of P. furiosus protein is weak. Probable reasons for these differences are discussed. Furthermore, unlike bacterial TruB and yeast Pus4, archaeal Pus10 does not require a U54*A58 reverse Hoogstein base pair and pyrimidine at position 56 to convert tRNA U55 to Psi55. The homology of eukaryal Pus10 with archaeal Pus10 suggests that the former may also have a tRNA Psi54 synthase activity.

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Evolution of Arabidopsis thaliana microRNAs from random sequences.

Publication Date: 2008 Oct 28 PMID: 18952822
Authors: Felippes, F. F. - Schneeberger, K. - Dezulian, T. - Huson, D. H. - Weigel, D.
Journal: RNA

One mechanism for the origin of new plant microRNAs (miRNAs) is from inverted duplications of transcribed genes. However, even though many young MIRNA genes have recently been identified in Arabidopsis thaliana, only a subset shows evidence for having evolved by this route. We propose that the hundreds of thousands of partially self-complementary foldback sequences found in a typical plant genome provide an alternative path for miRNA evolution. Our genome-wide analyses of young MIRNA genes suggest that some arose from DNA that either has self-complementarity by chance or that represents a highly eroded inverted duplication. These observations are compatible with the idea that, following capture of transcriptional regulatory sequences, random foldbacks can occasionally spawn new miRNAs. Subsequent stabilization through coevolution with initially fortuitous targets may lead to fixation of a small subset of these proto-miRNA genes.

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Thermodynamic examination of trinucleotide bulged RNA in the context of HIV-1 TAR RNA.

Publication Date: 2008 Oct 24 PMID: 18952821
Authors: Carter-O'Connell, I. - Booth, D. - Eason, B. - Grover, N.
Journal: RNA

RNA structures contain many bulges and loops that are expected to be sites for inter- and intra-molecular interactions. Nucleotides in the bulge are expected to influence the structure and recognition of RNA. The same stability is assigned to all trinucleotide bulged RNA in the current secondary structure prediction models. In this study thermal denaturation experiments were performed on four trinucleotide bulged RNA, in the context of HIV-1 TAR RNA, to determine whether the bulge sequence affects RNA stability and its divalent ion interactions. Cytosine-rich bulged RNA were more stable than uracil-rich bulged RNA in 1 M KCl. Interactions of divalent ions were more favorable with uracil-rich bulged RNA by approximately 2 kcal/mol over cytosine-rich bulged RNA. The UCU-TAR RNA (wild type) is stabilized by 1.7 kcal/mol in 9.5 mM Ca(2+) as compared with 1 M KCl, whereas no additional gain in stability is measured for CCC-TAR RNA. These results have implications for base substitution experiments traditionally employed to identify metal ion binding sites. To our knowledge, this is the first systematic study to quantify the effect of small sequence changes on RNA stability upon interactions with divalent ions.

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RNA target specificity of the embryonic cell fate determinant POS-1.

Publication Date: 2008 Oct 24 PMID: 18952820
Authors: Farley, B. M. - Pagano, J. M. - Ryder, S. P.
Journal: RNA

Specification of Caenorhabditis elegans body axes and cell fates occurs prior to the activation of zygotic transcription. Several CCCH-type tandem zinc finger (TZF) proteins coordinate local activation of quiescent maternal mRNAs after fertilization, leading to asymmetric expression of factors required for patterning. The primary determinant of posterior fate is the TZF protein POS-1. Mutants of pos-1 are maternal effect lethal with a terminal phenotype that includes excess pharyngeal tissue and no endoderm or germline. Here, we delineate the consensus POS-1 recognition element (PRE) required for specific recognition of its target mRNAs. The PRE is necessary but not sufficient to pattern the expression of a reporter. The PRE is distinct from sequences recognized by related proteins from both mammals and nematodes, demonstrating that variants of this protein family can recognize divergent RNA sequences. The PRE is found within the 3' untranslated region of 227 maternal transcripts required for early development, including genes involved in endoderm and germline specification. The results enable prediction of novel targets that explain the pleiotropy of the pos-1 phenotype.

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Flexibility in the site of exon junction complex deposition revealed by functional group and RNA secondary structure alterations in the splicing substrate.

Publication Date: 2008 Oct 24 PMID: 18952819
Authors: Mishler, D. M. - Christ, A. B. - Steitz, J. A.
Journal: RNA

The exon junction complex (EJC) is critical for mammalian nonsense-mediated mRNA decay and translational regulation, but the mechanism of its stable deposition on mRNA is unknown. To examine requirements for EJC deposition, we created splicing substrates containing either DNA nucleotides or RNA secondary structure in the 5' exon. Using RNase H protection, toeprinting, and coimmunoprecipitation assays, we found that EJC location shifts upstream when a stretch of DNA or RNA secondary structure appears at the canonical deposition site. These upstream shifts occur prior to exon ligation and are often accompanied by decreases in deposition efficiency. Although the EJC core protein eIF4AIII contacts four ribose 2'OH groups in crystal structures, we demonstrate that three 2'OH groups are sufficient for deposition. Thus, the site of EJC deposition is more flexible than previously appreciated and efficient deposition appears spatially limited.

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Suppressors of the cdc-25.1(gf)-associated intestinal hyperplasia reveal important maternal roles for prp-8 and a subset of splicing factors in C. elegans.

Publication Date: 2008 Oct 22 PMID: 18945809
Authors: Hebeisen, M. - Drysdale, J. - Roy, R.
Journal: RNA

The maternal contribution of gene products enables embryos to initiate their developmental program in the absence of zygotic gene expression. In Caenorhabditis elegans, maternal CDC-25.1 levels are tightly regulated to promote early cell divisions, while stabilization of this phosphatase by gain-of-function mutations gives rise to intestinal-specific hyperplasia. To identify regulators of CDC-25.1 levels and/or function, we performed a modifier screen of the cdc-25.1(gf)-dependent hyperplasia. One of the isolated suppressor mutants possesses a donor splice site mutation in prp-8, a key splicing factor of the U5-specific snRNP. prp-8(rr40) produces aberrant prp-8 splice variants that generate C-terminal truncations at the expense of wild-type prp-8. Levels of maternal transcripts are reduced, including cdc-25.1, while zygotic transcripts appear unperturbed, suggesting a germ-line-specific role for this splicing factor in regulating the splicing, and consequently, the steady-state levels of maternal transcripts. Using a novel feeding RNAi strategy we found that only a subset of splicing factors suppress cdc-25.1(gf), suggesting that they too may play specific roles in germ-line spliceosome function. In humans, mutations in the corresponding hPrp8 C-terminal domain result in retinitis pigmentosa, a retinal-specific disorder. Intriguingly, despite affecting the general splicing apparatus, both human and C. elegans show tissue-specific defects resulting from mutations in this key splicing component. Our findings suggest that in addition to its important regulatory function in the C. elegans germ line, prp-8(rr40) may provide further insight into the etiology of this splicing-associated human disorder.

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A mutant screen reveals RNase E as a silencer of group II intron retromobility in Escherichia coli.

Publication Date: 2008 Oct 22 PMID: 18945808
Authors: Coros, C. J. - Piazza, C. L. - Chalamcharla, V. R. - Belfort, M.
Journal: RNA

Group II introns are mobile retroelements that invade their hosts. The Lactococcus lactis group II intron recruits cellular polymerases, nucleases, and DNA ligase to complete the retromobility process in Escherichia coli. Here we describe a genetic screen with a Tn5 transposon library to identify other E. coli functions involved in retromobility of the L. lactis LtrB intron. Thirteen disruptions that reproducibly resulted in increased or decreased retrohoming levels into the E. coli chromosome were isolated. These functions were classified as factors involved in RNA processing, DNA replication, energy metabolism, and global regulation. Here we characterize a novel mutant in the rne promoter region, which regulates RNase E expression. Retrohoming and retrotransposition levels are elevated in the rneTn5 mutant. The stimulatory effect of the mutation on retromobility results from intron RNA accumulation in the RNase E mutant. These results suggest that RNase E, which is the central component of the RNA degradosome, could regulate retrohoming levels in response to cellular physiology.

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Intragenic rearrangements of a mycoreovirus induced by the multifunctional protein p29 encoded by the prototypic hypovirus CHV1-EP713.

Publication Date: 2008 Oct 22 PMID: 18945807
Authors: Sun, L. - Suzuki, N.
Journal: RNA

Mycoreovirus 1 (MyRV1), a member of the Reoviridae family possessing a genome consisting of 11 dsRNA segments (S1-S11), and the prototype hypovirus (CHV1-EP713) of the Hypoviridae family, which is closely related to the monopartite picorna-like superfamily with a ssRNA genome, infect the chestnut blight fungus and cause virulence attenuation and distinct phenotypic alterations in the host. Here, we present evidence for reproducible induction of intragenic rearrangements of MyRV1 S6 and S10, mediated by the multifunctional protein p29 encoded by CHV1. S6 and S10 underwent an almost full-length ORF duplication (S6L) and an internal deletion of three-fourths of the ORF (S10ss). No significant influence on symptom induction in the fungal host was associated with the S6L rearrangement. In contrast, S10-encoded VP10, while nonessential for MyRV1 replication, was shown to contribute to virulence reduction and reduced growth of aerial mycelia. Furthermore, p29 was found to copurify with MyRV1 genomic RNA and bind to VP9 in vitro and in vivo, suggesting direct interactions of p29 with the MyRV1 replication machinery. This study provides the first example of a viral factor involved in RNA genome rearrangements of a different virus and shows its usefulness as a probe into the mechanism of replication and symptom expression of a heterologous virus.

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The RNA WikiProject: Community annotation of RNA families.

Publication Date: 2008 Oct 22 PMID: 18945806
Authors: Daub, J. - Gardner, P. P. - Tate, J. - Ramskold, D. - Manske, M. - Scott, W. G. - Weinberg, Z. - Griffiths-Jones, S. - Bateman, A.
Journal: RNA

The online encyclopedia Wikipedia has become one of the most important online references in the world and has a substantial and growing scientific content. A search of Google with many RNA-related keywords identifies a Wikipedia article as the top hit. We believe that the RNA community has an important and timely opportunity to maximize the content and quality of RNA information in Wikipedia. To this end, we have formed the RNA WikiProject (http://en.wikipedia.org/wiki/Wikipedia:WikiProject_RNA) as part of the larger Molecular and Cellular Biology WikiProject. We have created over 600 new Wikipedia articles describing families of noncoding RNAs based on the Rfam database, and invite the community to update, edit, and correct these articles. The Rfam database now redistributes this Wikipedia content as the primary textual annotation of its RNA families. Users can, therefore, for the first time, directly edit the content of one of the major RNA databases. We believe that this Wikipedia/Rfam link acts as a functioning model for incorporating community annotation into molecular biology databases.

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Experimental identification of microRNA-140 targets by silencing and overexpressing miR-140.

Publication Date: 2008 Oct 22 PMID: 18945805
Authors: Nicolas, F. E. - Pais, H. - Schwach, F. - Lindow, M. - Kauppinen, S. - Moulton, V. - Dalmay, T.
Journal: RNA

MicroRNAs (miRNAs) are short noncoding RNA molecules regulating the expression of mRNAs. Target identification of miRNAs is computationally difficult due to the relatively low homology between miRNAs and their targets. We present here an experimental approach to target identification where the cartilage-specific miR-140 was overexpressed and silenced in cells it is normally expressed in separate experiments. Expression of mRNAs was profiled in both experiments and the intersection of mRNAs repressed by miR-140 overexpression and derepressed by silencing of miR-140 was identified. The intersection contained only 49 genes, although both treatments affected the accumulation of hundreds of mRNAs. These 49 genes showed a very strong enrichment for the miR-140 seed sequence implying that the approach is efficient and specific. 21 of these 49 genes were predicted to be direct targets based on the presence of the seed sequence. Interestingly, none of these were predicted by the published target prediction methods we used. One of the potential target mRNAs, Cxcl12, was experimentally validated by Northern blot analysis and a luciferase reporter assay.

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Stabilization of RNA during laser capture microdissection by performing experiments under argon atmosphere or using ethanol as a solvent in staining solutions.

Publication Date: 2008 Oct 22 PMID: 18945804
Authors: Clement-Ziza, M. - Munnich, A. - Lyonnet, S. - Jaubert, F. - Besmond, C.
Journal: RNA

The combination of laser capture microdissection (LCM) and gene expression experiments allows cell specific expression profiling, which is decisive in cellular transcriptomic exploration. LCM makes possible the isolation of unique cells or group of cells, but maintaining RNA quality during this process is challenging. Several protocols are available for section preparation, but none of those guarantees the integrity of the RNA during microdissection, and operators are recommended to perform LCM during a limited time. We hypothesized that the cause of RNA degradation during the microdissection time is the presence of water rendering endogenous RNase activity possible. We thus developed two methods that stabilize RNA during microdissection time for up to 90 min. The first one consists of performing LCM under an argon atmosphere, thus preventing tissue rehydration; it is compliant with all existing microdissection protocols. The second one is a new fixation and staining method using ethanol as solvent in all preparatory steps to LCM that enhances fixation and dehydration of samples. We assessed several stains in regard of their effect on tissue morphology and RNA integrity and adjusted an ethanolic staining solution of cresyl violet and eosin Y.

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Riboswitches in unexpected places--A synthetic riboswitch in a protein coding region.

Publication Date: 2008 Oct 22 PMID: 18945803
Authors: Topp, S. - Gallivan, J. P.
Journal: RNA

In natural and engineered systems, cis-RNA regulatory elements such as riboswitches are typically found within untranslated regions rather than within the protein coding sequences of genes. However, RNA sequences with important regulatory roles can exist within translated regions. Here, we present a synthetic riboswitch that is encoded within the translated region of a gene and represses Escherichia coli gene expression greater than 25-fold in the presence of a small-molecule ligand. The ability to encode riboswitches within translated regions as well as untranslated regions provides additional opportunities for creating new genetic control elements. Furthermore, evidence that a riboswitch can function in the translated region of a gene suggests that future efforts to identify natural riboswitches should consider this possibility.

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Exonucleolysis is required for nuclear mRNA quality control in yeast THO mutants.

Publication Date: 2008 Nov PMID: 18824516
Authors: Assenholt, J. - Mouaikel, J. - Andersen, K. R. - Brodersen, D. E. - Libri, D. - Jensen, T. H.
Journal: RNA

Production of aberrant messenger ribonucleoprotein particles (mRNPs) is subject to quality control (QC). In yeast strains carrying mutations of the THO complex, transcription induction triggers a number of interconnected QC phenotypes: (1) rapid degradation of several mRNAs; (2) retention of a fraction of THO-dependent mRNAs in transcription site-associated foci; and (3) formation of a high molecular weight DNA/protein complex in the 3'-ends of THO target genes. Here, we demonstrate that the 3'-5' exonucleolytic domain of the nuclear exosome factor Rrp6p is necessary for establishing all QC phenotypes associated with THO mutations. The N terminus of Rrp6p is also important presumably through its binding to the Rrp6p co-factor Rrp47p. Interestingly, the 3'-5' exonucleolytic activity of Dis3p, the only other active exonuclease of the nuclear exosome, can also contribute to RNA QC in THO mutants, while other nuclear 3'-5' exonucleases cannot. Our data show that exonucleolytic attack by the nuclear exosome is needed both for provoking mRNP QC and for its ensuing elimination of faulty RNA.

MeSH Categories: Exoribonucleases/genetics/*metabolism, Fungal Proteins/metabolism, Nuclear Proteins/metabolism, Protein Structure, Tertiary/genetics, *RNA Processing, Post-Transcriptional, RNA, Fungal/genetics/*metabolism, RNA, Messenger/genetics/*metabolism, Ribonucleoproteins/genetics/*metabolism, Saccharomyces cerevisiae/*enzymology/genetics, Saccharomyces cerevisiae Proteins/genetics/*metabolism, Sequence Deletion

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Polynucleotide phosphorylase hinders mRNA degradation upon ribosomal protein S1 overexpression in Escherichia coli.

Publication Date: 2008 Nov PMID: 18824515
Authors: Briani, F. - Curti, S. - Rossi, F. - Carzaniga, T. - Mauri, P. - Deho, G.
Journal: RNA

The exoribonuclease polynucleotide phosphorylase (PNPase, encoded by pnp) is a major player in bacterial RNA decay. In Escherichia coli, PNPase expression is post-transcriptionally regulated at the level of mRNA stability. The primary transcript is very efficiently processed by the endonuclease RNase III at a specific site and the processed pnp mRNA is rapidly degraded in a PNPase-dependent manner. While investigating the PNPase autoregulation mechanism we found, by UV-cross-linking experiments, that the ribosomal protein S1 in crude extracts binds to the pnp-mRNA leader region. We assayed the potential role of S1 protein in pnp gene regulation by modulating S1 expression from depletion to overexpression. We found that S1 depletion led to a sharp decrease of the amount of pnp and other tested mRNAs, as detected by Northern blotting, whereas S1 overexpression caused a strong stabilization of pnp and the other transcripts. Surprisingly, mRNA stabilization depended on PNPase, as it was not observed in a pnp deletion strain. PNPase-dependent stabilization, however, was not detected by chemical decay assay of bulk mRNA. Overall, our data suggest that PNPase exonucleolytic activity may be modulated by the translation potential of the target mRNAs and that, upon ribosomal protein S1 overexpression, PNPase protects from degradation a set of full-length mRNAs. It thus appears that a single mRNA species may be differentially targeted to either decay or PNPase-dependent stabilization, thus preventing its depletion in conditions of fast turnover.

MeSH Categories: Escherichia coli/*enzymology/genetics, Escherichia coli Proteins/genetics/*metabolism, Gene Expression Regulation, Bacterial, Gene Expression Regulation, Enzymologic, Polyribonucleotide Nucleotidyltransferase/genetics/*metabolism, Protein Binding, *RNA Stability, RNA, Bacterial/metabolism, RNA, Messenger/*metabolism, Ribosomal Proteins/genetics/*metabolism

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Kinetic and thermodynamic studies of peptidyltransferase in ribosomes from the extreme thermophile Thermus thermophilus.

Publication Date: 2008 Nov PMID: 18824514
Authors: Rodriguez-Correa, D. - Dahlberg, A. E.
Journal: RNA

Throughout evolution, emerging organisms survived by adapting existing biochemical processes to new reaction conditions. Simple protein enzymes balanced changes in structural stability with changes that permitted optimal catalysis by adjustments in both entropic and enthalpic contributions to the free energy of activation for the reaction. Study of adaptive mechanisms by large multicomponent enzymes such as the ribosome has been largely unexplored. Here we have determined the kinetic and thermodynamic parameters of peptidyltransferase in ribosomes from the extreme thermophile Thermus thermophilus. Activity of thermophilic enzymes can be assayed over a wide range of temperatures, enabling one to measure accurate catalytic rates and determine enthalpic and entropic contributions to the free energy of activation of the reaction. Differences in the reaction conditions used here and in published studies on mesophilic ribosomes prevent direct comparison, but our data on Thermus ribosomes suggest that these ribosomes have adapted to changing environments using the same strategies as simple protein enzymes, balancing stability and flexibility without loss of catalytic rate. This strategy must be a very ancient process, perhaps first used by primitive ribosomes in the RNA World.

MeSH Categories: Bacterial Proteins/*chemistry, Catalysis, Entropy, Heat, Peptides/chemistry, Peptidyl Transferases/*chemistry, RNA, Transfer, Met/chemistry, Ribosomes/chemistry/*enzymology, Thermus thermophilus/*enzymology

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Mutational analysis of the U12-dependent branch site consensus sequence.

Publication Date: 2008 Nov PMID: 18824513
Authors: Brock, J. E. - Dietrich, R. C. - Padgett, R. A.
Journal: RNA

Highly conserved sequences at the 5' splice site and branch site of U12-dependent introns are important determinants for splicing by U12-dependent spliceosomes. This study investigates the in vivo splicing phenotypes of mutations in the branch site consensus sequence of the U12-dependent intron F from a human NOL1 (P120) minigene. Intron F contains a fully consensus branch site sequence (UUCCUUAAC). Mutations at each position were analyzed for their effects on U12-dependent splicing in vivo. Mutations at most positions resulted in a significant reduction of correct U12-dependent splicing. Defects observed included increased unspliced RNA levels, the activation of cryptic U2-dependent 5' and 3' splice sites, and the activation of cryptic U12-dependent branch/3' splice sites. A strong correlation was observed between the predicted thermodynamic stability of the branch site: U12 snRNA interaction and correct U12-dependent splicing. The lack of a polypyrimidine tract between the branch site and 3' splice site of U12-dependent introns and the observed reliance on base-pairing interactions for correct U12-dependent splicing emphasize the importance of RNA/RNA interactions during U12-dependent intron recognition and proper splice site selection.

MeSH Categories: *Alternative Splicing, Base Sequence, Consensus Sequence, Humans, *Introns, Mutation, Nuclear Proteins/genetics, *RNA Splice Sites/genetics, RNA, Small Nuclear/genetics/*metabolism, Spliceosomes/genetics/metabolism

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The 3' proximal translational enhancer of Turnip crinkle virus binds to 60S ribosomal subunits.

Publication Date: 2008 Nov PMID: 18824512
Authors: Stupina, V. A. - Meskauskas, A. - McCormack, J. C. - Yingling, Y. G. - Shapiro, B. A. - Dinman, J. D. - Simon, A. E.
Journal: RNA

During cap-dependent translation of eukaryotic mRNAs, initiation factors interact with the 5' cap to attract ribosomes. When animal viruses translate in a cap-independent fashion, ribosomes assemble upstream of initiation codons at internal ribosome entry sites (IRES). In contrast, many plant viral genomes do not contain 5' ends with substantial IRES activity but instead have 3' translational enhancers that function by an unknown mechanism. A 393-nucleotide (nt) region that includes the entire 3' UTR of the Turnip crinkle virus (TCV) synergistically enhances translation of a reporter gene when associated with the TCV 5' UTR. The major enhancer activity was mapped to an internal region of approximately 140 nt that partially overlaps with a 100-nt structural domain previously predicted to adopt a form with some resemblance to a tRNA, according to a recent study by J.C. McCormack and colleagues. The T-shaped structure binds to 80S ribosomes and 60S ribosomal subunits, and binding is more efficient in the absence of surrounding sequences and in the presence of a pseudoknot that mimics the tRNA-acceptor stem. Untranslated TCV satellite RNA satC, which contains the TCV 3' end and 6-nt differences in the region corresponding to the T-shaped element, does not detectably bind to 80S ribosomes and is not predicted to form a comparable structure. Binding of the TCV T-shaped element by 80S ribosomes was unaffected by salt-washing, reduced in the presence of AcPhe-tRNA, which binds to the P-site, and enhanced binding of Phe-tRNA to the ribosome A site. Mutations that reduced translation in vivo had similar effects on ribosome binding in vitro. This strong correlation suggests that ribosome entry in the 3' UTR is a key function of the 3' translational enhancer of TCV and that the T-shaped element contains some tRNA-like properties.

MeSH Categories: 3' Untranslated Regions/genetics/*metabolism, Base Sequence, Carmovirus/*genetics, *Enhancer Elements (Genetics)/genetics, Genome, Viral, Molecular Sequence Data, Mutation, Protein Biosynthesis/*genetics, RNA, Transfer, Amino Acyl/metabolism, RNA, Viral/genetics/*metabolism, Ribosome Subunits, Large, Eukaryotic/*metabolism

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A novel biochemical method to identify target genes of individual microRNAs: identification of a new Caenorhabditis elegans let-7 target.

Publication Date: 2008 Nov PMID: 18824511
Authors: Andachi, Y.
Journal: RNA

MicroRNAs (miRNAs) are roughly 22-nucleotide regulatory RNAs that play important roles in many developmental and physiological processes. Animal miRNAs down-regulate target genes by forming imperfect base pairs with 3' untranslated regions (3' UTRs) of their mRNAs. Thousands of miRNAs have been discovered in several organisms. However, the target genes of almost all of these miRNAs remain to be identified. Here, we describe a method for isolating cDNA clones of target mRNAs that form base pairs in vivo with an endogenous miRNA of interest, in which the cDNAs are synthesized from the mRNAs using the miRNA as a reverse-transcription primer. The application of this method to Caenorhabditis elegans miRNA lin-4 under test conditions yielded many clones of the known target gene lin-14 that correspond to partial sequences 5' to lin-4 binding sites in the 3' UTR. The method was also applied to C. elegans miRNA let-7 and a new target gene responsible for the lethal phenotype in let-7 mutants was identified. These results demonstrate that the method is a useful way to identify targets on the basis of base pairing with individual miRNAs.

MeSH Categories: Animals, Base Sequence, Caenorhabditis elegans/*genetics, Cloning, Molecular, DNA, Complementary/genetics/*isolation & purification/metabolism, *Gene Expression Regulation, MicroRNAs/genetics/*metabolism, Molecular Sequence Data, Reverse Transcriptase Polymerase Chain Reaction/*methods

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Characterization of the termination-reinitiation strategy employed in the expression of influenza B virus BM2 protein.

Publication Date: 2008 Nov PMID: 18824510
Authors: Powell, M. L. - Napthine, S. - Jackson, R. J. - Brierley, I. - Brown, T. D.
Journal: RNA

Coupled expression of the M1 and BM2 open-reading frames (ORFs) of influenza B from the dicistronic segment 7 mRNA occurs by a process of termination-dependent reinitiation. The AUG start codon of the BM2 ORF overlaps the stop codon of the upstream M1 ORF in the pentanucleotide UAAUG, and BM2 synthesis is dependent upon translation of the M1 ORF and termination at the stop codon. Here, we have investigated the mRNA sequence requirements for BM2 expression. Termination-reinitiation is dependent upon 45 nucleotide (nt) of RNA immediately upstream of the UAAUG pentanucleotide, which includes an essential stretch complementary to 18S rRNA helix 26. Thus, similar to the caliciviruses, base-pairing between mRNA and rRNA is likely to play a role in tethering the 40S subunit to the mRNA following termination at the M1 stop codon. Consistent with this, repositioning of the M1 stop codon more than 24 nt downstream from the BM2 start codon inhibited BM2 expression. RNA structure probing revealed that the RNA upstream of the UAAUG overlap is not highly structured, but upon encountering the M1 stop codon by the ribosome, a stem-loop may form immediately 5' of the ribosome, with the 18S rRNA complementary region in the apical loop and in close proximity to helix 26. Mutational analysis reveals that the normal requirements for start site selection in BM2 expression are suspended, with little effect of initiation codon context and efficient use of noncanonical initiation codons. This suggests that the full complement of initiation factors is not required for the reinitiation process.

MeSH Categories: Amino Acid Sequence, Base Sequence, Codon, Initiator/genetics/metabolism, Codon, Terminator/genetics/metabolism, Influenza B virus/genetics/*metabolism, Models, Biological, Molecular Sequence Data, Mutation, Nucleic Acid Conformation, *Peptide Chain Initiation, Translational/genetics, *Peptide Chain Termination, Translational/genetics, RNA, Messenger/chemistry/genetics/metabolism, RNA, Ribosomal, 18S/chemistry/genetics/metabolism, RNA, Viral/chemistry/genetics/metabolism, Viral Proteins/*biosynthesis/genetics

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Analysis and classification of RNA tertiary structures.

Publication Date: 2008 Nov PMID: 18824509
Authors: Abraham, M. - Dror, O. - Nussinov, R. - Wolfson, H. J.
Journal: RNA

There is a fast growing interest in noncoding RNA transcripts. These transcripts are not translated into proteins, but play essential roles in many cellular and pathological processes. Recent efforts toward comprehension of their function has led to a substantial increase in both the number and the size of solved RNA structures. With the aim of addressing questions relating to RNA structural diversity, we examined RNA conservation at three structural levels: primary, secondary, and tertiary structure. Additionally, we developed an automated method for classifying RNA structures based on spatial (three-dimensional [3D]) similarity. Applying the method to all solved RNA structures resulted in a classified database of RNA tertiary structures (DARTS). DARTS embodies 1333 solved RNA structures classified into 94 clusters. The classification is hierarchical, reflecting the structural relationship between and within clusters. We also developed an application for searching DARTS with a new structure. The search is fast and its performance was successfully tested on all solved RNA structures since the creation of DARTS. A user-friendly interface for both the database and the search application is available online. We show intracluster and intercluster similarities in DARTS and demonstrate the usefulness of the search application. The analysis reveals the current structural repertoire of RNA and exposes common global folds and local tertiary motifs. Further study of these conserved substructures may suggest possible RNA domains and building blocks. This should be beneficial for structure prediction and for gaining insights into structure-function relationships.

MeSH Categories: Alternative Splicing, Base Sequence, Computational Biology/*methods, Conserved Sequence, Databases, Nucleic Acid, Introns, Molecular Sequence Data, *Nucleic Acid Conformation, RNA, Ribosomal, 5S/chemistry/classification, RNA, Untranslated/*chemistry/*classification, Sequence Analysis, RNA/*methods

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Peptidyl-CCA deacylation on the ribosome promoted by induced fit and the O3'-hydroxyl group of A76 of the unacylated A-site tRNA.

Publication Date: 2008 Nov PMID: 18818369
Authors: Simonovic, M. - Steitz, T. A.
Journal: RNA

The last step in ribosome-catalyzed protein synthesis is the hydrolytic release of the newly formed polypeptide from the P-site bound tRNA. Hydrolysis of the ester link of the peptidyl-tRNA is stimulated normally by the binding of release factors (RFs). However, an unacylated tRNA or just CCA binding to the ribosomal A site can also stimulate deacylation under some nonphysiological conditions. Although the sequence of events is well described by biochemical studies, the structural basis of the mechanism underlying this process is not well understood. Two new structures of the large ribosomal subunit of Haloarcula marismortui complexed with a peptidyl-tRNA analog in the P site and two oligonucleotide mimics of unacylated tRNA, CCA and CA, in the A site show that the binding of either CA or CCA induces a very similar conformational change in the peptidyl-transferase center as induced by aminoacyl-CCA. However, only CCA positions a water molecule appropriately to attack the carbonyl carbon of the peptidyl-tRNA and stabilizes the proper orientation of the ester link for hydrolysis. We, thus, conclude that both the ability of the O3'-hydroxyl group of the A-site A76 to position the water and the A-site CCA induced conformational change of the PTC are critical for the catalysis of the deacylation of the peptidyl-tRNA by CCA, and perhaps, an analogous mechanism is used by RFs.

MeSH Categories: Acylation, Binding Sites, Catalysis, Crystallography, Dinucleoside Phosphates/chemistry/metabolism, Haloarcula/metabolism, Hydrolysis, Nucleic Acid Conformation, Oligonucleotides/chemistry, *Protein Biosynthesis, RNA, Transfer, Amino Acyl/chemistry/*metabolism, Ribosome Subunits, Large, Archaeal/chemistry/*metabolism, Water/chemistry

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MicroRNA miR-21 overexpression in human breast cancer is associated with advanced clinical stage, lymph node metastasis and patient poor prognosis.

Publication Date: 2008 Nov PMID: 18812439
Authors: Yan, L. X. - Huang, X. F. - Shao, Q. - Huang, M. Y. - Deng, L. - Wu, Q. L. - Zeng, Y. X. - Shao, J. Y.
Journal: RNA

To investigate the global expression profile of miRNAs in primary breast cancer (BC) and normal adjacent tumor tissues (NATs) and its potential relevance to clinicopathological characteristics and patient survival, the genome-wide expression profiling of miRNAs in BC was investigated using a microarray containing 435 mature human miRNA oligonucleotide probes. Nine miRNAs of hsa-miR-21, hsa-miR-365, hsa-miR-181b, hsa-let-7f, hsa-miR-155, hsa-miR-29b, hsa-miR-181d, hsa-miR-98, and hsa-miR-29c were observed to be up-regulated greater than twofold in BC compared with NAT, whereas seven miRNAs of hsa-miR-497, hsa-miR-31, hsa-miR-355, hsa-miR-320, rno-mir-140, hsa-miR-127 and hsa-miR-30a-3p were observed to be down-regulated greater than twofold. The most significantly up-regulated miRNAs, hsa-mir-21 (miR-21), was quantitatively analyzed by TaqMan real-time PCR in 113 BC tumors. Interestingly, among the 113 BC cases, high level expression of miR-21 was significantly correlated with advanced clinical stage (P = 0.006, Fisher's exact text), lymph node metastasis (P = 0.007, Fisher's exact text), and shortened survival of the patients (hazard ratio [HR]=5.476, P < 0.001). Multivariate Cox regression analysis revealed this prognostic impact (HR=4.133, P = 0.001) to be independent of disease stage (HR=2.226, P = 0.013) and histological grade (HR=3.681, P = 0.033). This study could identify the differentiated miRNAs expression profile in BC and reveal that miR-21 overexpression was correlated with specific breast cancer biopathologic features, such as advanced tumor stage, lymph node metastasis, and poor survival of the patients, indicating that miR-21 may serve as a molecular prognostic marker for BC and disease progression.

MeSH Categories: Adult, Aged, Breast Neoplasms/*genetics/*pathology, Female, Gene Expression Profiling, Gene Expression Regulation, Neoplastic, Humans, Lymphatic Metastasis, MicroRNAs/*genetics, Middle Aged, Prognosis, Tumor Markers, Biological/*genetics

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Crystal structure of Escherichia coli PNPase: central channel residues are involved in processive RNA degradation.

Publication Date: 2008 Nov PMID: 18812438
Authors: Shi, Z. - Yang, W. Z. - Lin-Chao, S. - Chak, K. F. - Yuan, H. S.
Journal: RNA

Bacterial polynucleotide phosphorylase (PNPase) plays a major role in mRNA turnover by the degradation of RNA from the 3'- to 5'-ends. Here, we determined the crystal structures of the wild-type and a C-terminal KH/S1 domain-truncated mutant (DeltaKH/S1) of Escherichia coli PNPase at resolutions of 2.6 A and 2.8 A, respectively. The six RNase PH domains of the trimeric PNPase assemble into a ring-like structure containing a central channel. The truncated mutant DeltaKH/S1 bound and cleaved RNA less efficiently with an eightfold reduced binding affinity. Thermal melting and acid-induced trimer dissociation studies, analyzed by circular dichroism and dynamic light scattering, further showed that DeltaKH/S1 formed a less stable trimer than the full-length PNPase. The crystal structure of DeltaKH/S1 is more expanded, containing a slightly wider central channel than that of the wild-type PNPase, suggesting that the KH/S1 domain helps PNPase to assemble into a more compact trimer, and it regulates the channel size allosterically. Moreover, site-directed mutagenesis of several arginine residues in the channel neck regions produced defective PNPases that either bound and cleaved RNA less efficiently or generated longer cleaved oligonucleotide products, indicating that these arginines were involved in RNA binding and processive degradation. Taking these results together, we conclude that the constricted central channel and the basic-charged residues in the channel necks of PNPase play crucial roles in trapping RNA for processive exonucleolytic degradation.

MeSH Categories: Amino Acid Sequence, Arginine/chemistry/genetics, Crystallography, Escherichia coli/*enzymology, Escherichia coli Proteins/*chemistry/genetics/metabolism, Molecular Sequence Data, Polyribonucleotide Nucleotidyltransferase/*chemistry/genetics/metabolism, Protein Structure, Tertiary, RNA Processing, Post-Transcriptional, RNA Stability/genetics, RNA, Messenger/chemistry/metabolism

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A bioinformatics tool for linking gene expression profiling results with public databases of microRNA target predictions.

Publication Date: 2008 Nov PMID: 18812437
Authors: Creighton, C. J. - Nagaraja, A. K. - Hanash, S. M. - Matzuk, M. M. - Gunaratne, P. H.
Journal: RNA

MicroRNAs are short (approximately 22 nucleotides) noncoding RNAs that regulate the stability and translation of mRNA targets. A number of computational algorithms have been developed to help predict which microRNAs are likely to regulate which genes. Gene expression profiling of biological systems where microRNAs might be active can yield hundreds of differentially expressed genes. The commonly used public microRNA target prediction databases facilitate gene-by-gene searches. However, integration of microRNA-mRNA target predictions with gene expression data on a large scale using these databases is currently cumbersome and time consuming for many researchers. We have developed a desktop software application which, for a given target prediction database, retrieves all microRNA:mRNA functional pairs represented by an experimentally derived set of genes. Furthermore, for each microRNA, the software computes an enrichment statistic for overrepresentation of predicted targets within the gene set, which could help to implicate roles for specific microRNAs and microRNA-regulated genes in the system under study. Currently, the software supports searching of results from PicTar, TargetScan, and miRanda algorithms. In addition, the software can accept any user-defined set of gene-to-class associations for searching, which can include the results of other target prediction algorithms, as well as gene annotation or gene-to-pathway associations. A search (using our software) of genes transcriptionally regulated in vitro by estrogen in breast cancer uncovered numerous targeting associations for specific microRNAs-above what could be observed in randomly generated gene lists-suggesting a role for microRNAs in mediating the estrogen response. The software and Excel VBA source code are freely available at http://sigterms.sourceforge.net.

MeSH Categories: Algorithms, Computational Biology/*methods, Databases, Nucleic Acid, *Gene Expression Profiling, MicroRNAs/*genetics, Sequence Analysis, RNA/*methods, *Software

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Distinct roles for Khd1p in the localization and expression of bud-localized mRNAs in yeast.

Publication Date: 2008 Nov PMID: 18805955
Authors: Hasegawa, Y. - Irie, K. - Gerber, A. P.
Journal: RNA

The RNA-binding protein Khd1p (KH-domain protein 1) is required for efficient localization of ASH1 mRNA to the bud-tip, probably acting as a translational repressor during mRNA transport in yeast. Here, we have systematically examined Khd1p mRNA targets and colocalization with known bud-tip-localized mRNAs in vivo. Affinity purification and DNA microarray analysis of Khd1p-associated mRNAs revealed hundreds of potential mRNAs targets, many of them encoding membrane-associated proteins. The putative targets include the messages for MID2, MTL1, WSC2, SRL1, EGT2, CLB2, ASH1, and Khd1p colocalizes with these mRNAs at the bud-tip. The combination of bioinformatics, RNA localization, and in vitro RNA-binding assays revealed that Khd1p binds to CNN repeats in coding regions of mRNA targets. Among the proteins encoded by previously known bud-tip-localized mRNAs, only Mtl1p levels were decreased in khd1Delta mutant cells, whereas Ash1p and Srl1p were reduced in cells overexpressing KHD1. Hence, Khd1p differentially affects gene expression possibly due to combinatorial arrangement with additional factors reflecting the redundant structure of post-transcriptional regulatory systems.

MeSH Categories: Amino Acid Sequence, Calcium-Binding Proteins/genetics, DNA-Binding Proteins/genetics, *Gene Expression Regulation, Fungal, Membrane Proteins/genetics, Molecular Sequence Data, Mutation, RNA, Fungal/*metabolism, RNA, Messenger/*metabolism, RNA-Binding Proteins/genetics/*metabolism, Repressor Proteins/genetics, Saccharomyces cerevisiae/genetics/*metabolism, Saccharomyces cerevisiae Proteins/genetics/*metabolism, Transcription, Genetic

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Mutational analysis of vaccinia virus mRNA cap (guanine-N7) methyltransferase reveals essential contributions of the N-terminal peptide that closes over the active site.

Publication Date: 2008 Nov PMID: 18799596
Authors: Zheng, S. - Shuman, S.
Journal: RNA

RNA guanine-N7 methyltransferase catalyzes the third step of eukaryal mRNA capping, the transfer of a methyl group from AdoMet to GpppRNA to form m(7)GpppRNA. Mutational and crystallographic analyses of cellular and poxvirus cap methyltransferases have yielded a coherent picture of a conserved active site and determinants of substrate specificity. Models of the Michaelis complex suggest a direct in-line mechanism of methyl transfer. Because no protein contacts to the guanine-N7 nucleophile, the AdoMet methyl carbon (Cepsilon) or the AdoHcy sulfur (Sdelta) leaving group were observed in ligand-bound structures of cellular cap methyltransferase, it was initially thought that the enzyme facilitates catalysis by optimizing proximity and geometry of the donor and acceptor. However, the structure of AdoHcy-bound vaccinia virus cap methyltransferase revealed the presence of an N-terminal "lid peptide" that closes over the active site and makes multiple contacts with the substrates, including the AdoMet sulfonium. This segment is disordered in the vaccinia apoenzyme and is not visible in the available structures of cellular cap methyltransferase. Here, we conducted a mutational analysis of the vaccinia virus lid peptide ((545)DKFRLNPEVSYFTNKRTRG(563)) entailing in vivo and in vitro readouts of the effects of alanine and conservative substitutions. We thereby identified essential functional groups that interact with the AdoMet sulfonium (Tyr555, Phe556), the AdoMet adenine (Asn550), and the cap triphosphate bridge (Arg560, Arg562). The results suggest that van der Waals contacts of Tyr555 and Phe556 to the AdoMet Sdelta and C epsilon atoms, and the electron-rich environment around the sulfonium, serve to stabilize the transition state of the transmethylation reaction.

MeSH Categories: Alanine/chemistry/genetics, Amino Acid Sequence, Binding Sites/genetics, Conserved Sequence, DNA Mutational Analysis, Methylation, Methyltransferases/chemistry/*genetics, Molecular Sequence Data, Multienzyme Complexes/chemistry/*genetics, Nucleotidyltransferases/chemistry/*genetics, Peptides/chemistry/genetics, Phenylalanine/chemistry/genetics, Phosphoric Monoester Hydrolases/chemistry/*genetics, Structure-Activity Relationship, Tyrosine/chemistry/genetics, Vaccinia virus/enzymology/*genetics

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Two CRM protein subfamilies cooperate in the splicing of group IIB introns in chloroplasts.

Publication Date: 2008 Nov PMID: 18799595
Authors: Asakura, Y. - Bayraktar, O. A. - Barkan, A.
Journal: RNA

Chloroplast genomes in angiosperms encode approximately 20 group II introns, approximately half of which are classified as subgroup IIB. The splicing of all but one of the subgroup IIB introns requires a heterodimer containing the peptidyl-tRNA hydrolase homolog CRS2 and one of two closely related proteins, CAF1 or CAF2, that harbor a recently recognized RNA binding domain called the CRM domain. Two CRS2/CAF-dependent introns require, in addition, a CRM domain protein called CFM2 that is only distantly related to CAF1 and CAF2. Here, we show that CFM3, a close relative of CFM2, associates in vivo with those CRS2/CAF-dependent introns that are not CFM2 ligands. Mutant phenotypes in rice and Arabidopsis support a role for CFM3 in the splicing of most of the introns with which it associates. These results show that either CAF1 or CAF2 and either CFM2 or CFM3 simultaneously bind most chloroplast subgroup IIB introns in vivo, and that the CAF and CFM subunits play nonredundant roles in splicing. These results suggest that the expansion of the CRM protein family in plants resulted in two subfamilies that play different roles in group II intron splicing, with further diversification within a subfamily to accommodate multiple intron ligands.

MeSH Categories: Arabidopsis/genetics/growth & development/metabolism, Arabidopsis Proteins/classification/genetics/metabolism, Chloroplasts/*genetics/metabolism, *Introns, Mitochondria, NADH Dehydrogenase/genetics, Oryza sativa/metabolism, Plant Proteins/classification/genetics/*metabolism, *RNA Splicing, Seeds/genetics/growth & development/metabolism, Zea mays/metabolism

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The effect of intron length on exon creation ratios during the evolution of mammalian genomes.

Publication Date: 2008 Nov PMID: 18796579
Authors: Roy, M. - Kim, N. - Xing, Y. - Lee, C.
Journal: RNA

Recent studies report that alternatively spliced exons tend to occur in longer introns, which is attributed to the length constraints for splice site pairing for the two major splicing mechanisms, intron definition versus exon definition. Using genome-wide studies of EST and microarray data from human and mouse, we have analyzed the distribution of various subsets of alternatively spliced exons, based on their inclusion level and evolutionary history, versus increasing intron length. Alternative exons may be included in either a major or minor fraction of all transcripts (known as major-form and minor-form exons, respectively). We find that major-form exons are seven- to eightfold more likely to be contained in short introns (<400 nt) than minor-form exons, which occur preferentially in longer introns. Since minor-form exons are more likely to be novel (approximately 75%), this implied that novel exons arise more frequently in longer introns. To test this hypothesis, we used whole genome alignments to classify exons according to their phylogenetic age. We find that older exons, i.e., exons that are conserved in all mammals, predominate at shorter intron lengths, for both major- and minor-form exons. In contrast, exons that arose recently during primate evolution are more prevalent at longer intron lengths (>1000 nt). This suggests that the observed correlation of longer intron lengths with alternatively spliced exons may be at least partly due to biases in the probability of exon creation, which is higher in long introns.

MeSH Categories: *Alternative Splicing, Animals, Computational Biology, Conserved Sequence, *Evolution, Molecular, *Exons, Expressed Sequence Tags, *Genome, Human, Humans, *Introns, Mice, Oligonucleotide Array Sequence Analysis, Phylogeny, RNA Splice Sites, Sequence Alignment, Sequence Analysis, DNA

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Overexpressed mitochondrial leucyl-tRNA synthetase suppresses the A3243G mutation in the mitochondrial tRNA(Leu(UUR)) gene.

Publication Date: 2008 Nov PMID: 18796578
Authors: Park, H. - Davidson, E. - King, M. P.
Journal: RNA

The A3243G mutation in the human mitochondrial tRNA(Leu(UUR)) gene causes a number of human diseases. This mutation reduces the level and fraction of aminoacylated tRNA(Leu(UUR)) and eliminates nucleotide modification at the wobble position of the anticodon. These deficiencies are associated with mitochondrial translation defects that result in decreased levels of mitochondrial translation products and respiratory chain enzyme activities. We have suppressed the respiratory chain defects in A3243G mutant cells by overexpressing human mitochondrial leucyl-tRNA synthetase. The rates of oxygen consumption in suppressed cells were directly proportional to the levels of leucyl-tRNA synthetase. Fifteenfold higher levels of leucyl-tRNA synthetase resulted in wild-type respiratory chain function. The suppressed cells had increased steady-state levels of tRNA(Leu(UUR)) and up to threefold higher steady-state levels of mitochondrial translation products, but did not have rates of protein synthesis above those in parental mutant cells. These data suggest that suppression of the A3243G mutation occurred by increasing protein stability. This suppression of a tRNA gene mutation by increasing the steady-state levels of its cognate aminoacyl-tRNA synthetase is a model for potential therapies for human pathogenic tRNA mutations.

MeSH Categories: Cell Line, DNA, Mitochondrial/genetics/metabolism, Electron Transport/genetics, Humans, Leucine-tRNA Ligase/*biosynthesis/genetics, Mitochondria/enzymology, Mitochondrial Diseases/*genetics, Mutation, Protein Biosynthesis/genetics, RNA, Transfer, Amino Acyl/*genetics/metabolism, *Suppression, Genetic

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