RNA

Dynamic isomiR regulation in Drosophila development.

Publication Date: 2010 Aug 30 PMID: 20805289
Authors: Fernandez-Valverde, S. L. - Taft, R. J. - Mattick, J. S.
Journal: RNA

Several recent reports have demonstrated that microRNAs (miRNAs) can exhibit heterogeneous ends and post-transcriptional nontemplate 3' end additions of uridines or adenosines. Using two small RNA deep-sequencing data sets, we show here that these miRNA isoforms (isomiRs) are differentially expressed across Drosophila melanogaster development and tissues. Specifically, we demonstrate that: (1) nontemplate nucleotide additions of adenosines to miRNA 3' ends are highly abundant in early development; (2) a subset of miRNAs with nontemplate 3' Us are expressed in adult tissues; and (3) the size of at least eight "mature" (unmodified) miRNAs varies in a life-cycle or tissue-specific manner. These results suggest that subtle variability in isomiR expression, which is widely thought to be the result of inexact Dicer processing, is regulated and biologically meaningful. Indeed, a subset of the miRNAs enriched for 3' adenosine additions during early embryonic development, including miR-282 and miR-312, show enrichment for target sites in developmental genes that are expressed during late embryogenesis, suggesting that nontemplate additions increase miRNA stability or strengthen miRNA:target interactions. This work suggests that isomiR expression is an important aspect of miRNA biology, which warrants further investigation.

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ncRNAimprint: A comprehensive database of mammalian imprinted noncoding RNAs.

Publication Date: 2010 Aug 26 PMID: 20801769
Authors: Zhang, Y. - Guan, D. G. - Yang, J. H. - Shao, P. - Zhou, H. - Qu, L. H.
Journal: RNA

Imprinted noncoding RNAs (ncRNAs) are expressed mono-allelically in a parent-of-origin-dependent manner, which is mainly evident in mammals. Lying at a crossroad between imprinted genes and ncRNAs, imprinted ncRNAs show distinct features. They are likely to function in nontraditional ways compared to non-imprinted ncRNAs, and are much more responsible for the mechanism of genomic imprinting compared to imprinted protein-coding genes. An increasing number of human diseases have been shown to be related to abnormalities in imprinted ncRNAs. Due to their functional importance, many studies focusing on imprinted ncRNAs have been published in recent years; however, there is no systematic collection or description of imprinted ncRNAs and the rapidly growing knowledge is scattered in various places. Here, we describe a new database, ncRNAimprint, which serves as a comprehensive resource center for mammalian imprinted ncRNAs. A catalog of imprinted ncRNAs, including snoRNAs, microRNAs, piRNAs, siRNAs, antisense ncRNAs, and mRNA-like ncRNAs, was annotated in detail using information extracted from relevant literature and databases. Comprehensive collections of imprinted ncRNA-related diseases, imprinting control regions (ICRs), and imprinted regions were manually compiled to provide resources for current research focusing on imprinted ncRNAs. Small RNA deep sequencing reads that fully matched within imprinted regions were also included to offer useful evidence in detecting novel imprinted ncRNAs and to aid in analyzing expression patterns of known imprinted ncRNAs. A search page including four effective search forms and two graphical browsers was created for rapid retrieval and visualization of these data. The imprinted ncRNA database is freely accessible at http://rnaqueen.sysu.edu.cn/ncRNAimprint.

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RPL30 regulation of splicing reveals distinct roles for Cbp80 in U1 and U2 snRNP cotranscriptional recruitment.

Publication Date: 2010 Aug 27 PMID: 20801768
Authors: Bragulat, M. - Meyer, M. - Macias, S. - Camats, M. - Labrador, M. - Vilardell, J.
Journal: RNA

Pre-mRNA splicing is catalyzed by the spliceosome, and its control is essential for correct gene expression. While splicing repressors typically interfere with transcript recognition by spliceosomal components, the yeast protein L30 blocks spliceosomal rearrangements required for the engagement of U2 snRNP (small ribonucleoprotein particle) to its own transcript RPL30. Using a mutation in the RPL30 binding site that disrupts this repression, we have taken a genetic approach to reveal that regulation of splicing is restored in this mutant by deletion of the cap-binding complex (CBC) component Cbp80. Indeed, our data indicate that Cbp80 plays distinct roles in the recognition of the intron by U1 and U2 snRNP. It promotes the initial 5' splice site recognition by U1 and, independently, facilitates U2 recruitment, depending on sequences located in the vicinity of the 5' splice site. These results reveal a novel function for CBC in splicing and imply that these molecular events can be the target of a splicing regulator.

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Functional elucidation of a key contact between tRNA and the large ribosomal subunit rRNA during decoding.

Publication Date: 2010 Aug 25 PMID: 20739608
Authors: Ortiz-Meoz, R. F. - Green, R.
Journal: RNA

The selection of cognate tRNAs during translation is specified by a kinetic discrimination mechanism driven by distinct structural states of the ribosome. While the biochemical steps that drive the tRNA selection process have been carefully documented, it remains unclear how recognition of matched codon:anticodon helices in the small subunit facilitate global rearrangements in the ribosome complex that efficiently promote tRNA decoding. Here we use an in vitro selection approach to isolate tRNA(Trp) miscoding variants that exhibit a globally perturbed tRNA tertiary structure. Interestingly, the most substantial distortions are positioned in the elbow region of the tRNA that closely approaches helix 69 (H69) of the large ribosomal subunit. The importance of these specific interactions to tRNA selection is underscored by our kinetic analysis of both tRNA and rRNA variants that perturb the integrity of this interaction.

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Differential assembly of 16S rRNA domains during 30S subunit formation.

Publication Date: 2010 Aug 24 PMID: 20736336
Authors: Xu, Z. - Culver, G. M.
Journal: RNA

Rapid and accurate assembly of the ribosomal subunits, which are responsible for protein synthesis, is required to sustain cell growth. Our best understanding of the interaction of 30S ribosomal subunit components (16S ribosomal RNA [rRNA] and 20 ribosomal proteins [r-proteins]) comes from in vitro work using Escherichia coli ribosomal components. However, detailed information regarding the essential elements involved in the assembly of 30S subunits still remains elusive. Here, we defined a set of rRNA nucleotides that are critical for the assembly of the small ribosomal subunit in E. coli. Using an RNA modification interference approach, we identified 54 nucleotides in 16S rRNA whose modification prevents the formation of a functional small ribosomal subunit. The majority of these nucleotides are located in the head and interdomain junction of the 30S subunit, suggesting that these regions are critical for small subunit assembly. In vivo analysis of specific identified sites, using engineered mutations in 16S rRNA, revealed defective protein synthesis capability, aberrant polysome profiles, and abnormal 16S rRNA processing, indicating the importance of these residues in vivo. These studies reveal that specific segments of 16S rRNA are more critical for small subunit assembly than others, and suggest a hierarchy of importance.

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Probing tRNA interaction with biogenic polyamines.

Publication Date: 2010 Aug 23 PMID: 20729276
Authors: Ouameur, A. A. - Bourassa, P. - Tajmir-Riahi, H. A.
Journal: RNA

Biogenic polyamines are found to modulate protein synthesis at different levels. This effect may be explained by the ability of polyamines to bind and influence the secondary structure of tRNA, mRNA, and rRNA. We report the interaction between tRNA and the three biogenic polyamines putrescine, spermidine, spermine, and cobalt(III)hexamine at physiological conditions, using FTIR spectroscopy, capillary electrophoresis, and molecular modeling. The results indicated that tRNA was stabilized at low biogenic polyamine concentration, as a consequence of polyamine interaction with the backbone phosphate group. The main tRNA reactive sites for biogenic polyamine at low concentration were guanine-N7/O6, uracil-O2/O4, adenine-N3, and 2'OH of the ribose. At high polyamine concentration, the interaction involves guanine-N7/O6, adenine-N7, uracil-O2 reactive sites, and the backbone phosphate group. The participation of the polycation primary amino group, in the interaction and the presence of the hydrophobic contact, are also shown. The binding affinity of biogenic polyamine to tRNA molecule was in the order of spermine > spermidine > putrescine with K (Spm) = 8.7 x 10(5) M(-1), K (Spd) = 6.1 x 10(5) M(-1), and K (Put) = 1.0 x 10(5) M(-1), which correlates with their positively charged amino group content. Hill analysis showed positive cooperativity for the biogenic polyamines and negative cooperativity for cobalt-hexamine. Cobalt(III)hexamine contains high- and low-affinity sites in tRNA with K (1) = 3.2 x 10(5) M(-1) and K (2) = 1.7 x 10(5) M(-1), that have been attributed to the interactions with guanine-N7 sites and the backbone PO(2) group, respectively. This mechanism of tRNA binding could explain the condensation phenomenon observed at high Co(III) content, as previously shown in the Co(III)-DNA complexes.

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Kinetic basis for global loss of fidelity arising from mismatches in the P-site codon:anticodon helix.

Publication Date: 2010 Aug 19 PMID: 20724456
Authors: Zaher, H. S. - Green, R.
Journal: RNA

Faithful decoding of the genetic information by the ribosome relies on kinetically driven mechanisms that promote selection of cognate substrates during elongation. Recently, we have shown that in addition to these kinetically driven mechanisms, the ribosome possesses a post peptidyl transfer quality control system that retrospectively monitors the codon-anticodon interaction in the P site, triggering substantial losses in the specificity of the A site during subsequent tRNA and RF selection when a mistake has occurred. Here, we report a detailed kinetic analysis of tRNA selection in the context of a mismatched P-site codon:anticodon interaction. We observe pleiotropic effects of a P-site mismatch on tRNA selection, such that near-cognate tRNA is processed by the ribosome almost as efficiently as cognate. In particular, after a miscoding event, near-cognate codon-anticodon complexes are stabilized on the ribosome to an extent similar to that observed for cognate ones. Moreover, the two observed forward rates of GTPase activation and accommodation are greatly accelerated ( approximately 10-fold) for near-cognate tRNAs. Because the ensemble of effects of a mismatched P site on substrate selection were found to be different from those reported for other ribosomal perturbations and miscoding agents, we propose that the structural integrity of the mRNA-tRNA helix in the P site provides a distinct molecular switch that dictates the specificity of the A site.

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Regulatory RNAs derived from transfer RNA?

Publication Date: 2010 Aug 18 PMID: 20719919
Authors: Pederson, T.
Journal: RNA

Four recent studies suggest that cleavages of transfer RNAs generate products with microRNA-like features, with some evidence of function. If their regulatory functions were to be confirmed, these newly revealed RNAs would add to the expanding repertoire of small noncoding RNAs and would also provide new perspectives on the coevolution of transfer RNA and messenger RNA.

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Subribosomal particle analysis reveals the stages of bacterial ribosome assembly at which rRNA nucleotides are modified.

Publication Date: 2010 Aug 18 PMID: 20719918
Authors: Siibak, T. - Remme, J.
Journal: RNA

Modified nucleosides of ribosomal RNA are synthesized during ribosome assembly. In bacteria, each modification is made by a specialized enzyme. In vitro studies have shown that some enzymes need the presence of ribosomal proteins while other enzymes can modify only protein-free rRNA. We have analyzed the addition of modified nucleosides to rRNA during ribosome assembly. Accumulation of incompletely assembled ribosomal particles (25S, 35S, and 45S) was induced by chloramphenicol or erythromycin in an exponentially growing Escherichia coli culture. Incompletely assembled ribosomal particles were isolated from drug-treated and free 30S and 50S subunits and mature 70S ribosomes from untreated cells. Nucleosides of 16S and 23S rRNA were prepared and analyzed by reverse-phase, high-performance liquid chromatography (HPLC). Pseudouridines were identified by the chemical modification/primer extension method. Based on the results, the rRNA modifications were divided into three major groups: early, intermediate, and late assembly specific modifications. Seven out of 11 modified nucleosides of 16S rRNA were late assembly specific. In contrast, 16 out of 25 modified nucleosides of 23S rRNA were made during early steps of ribosome assembly. Free subunits of exponentially growing bacteria contain undermodified rRNA, indicating that a specific set of modifications is synthesized during very late steps of ribosome subunit assembly.

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The adaptive significance of unproductive alternative splicing in primates.

Publication Date: 2010 Aug 18 PMID: 20719917
Authors: Skandalis, A. - Frampton, M. - Seger, J. - Richards, M. H.
Journal: RNA

Alternative gene splicing is pervasive in metazoa, particularly in humans, where the majority of genes generate splice variant transcripts. Characterizing the biological significance of alternative transcripts is methodologically difficult since it is impractical to assess thousands of splice variants as to whether they actually encode proteins, whether these proteins are functional, or whether transcripts have a function independent of protein synthesis. Consequently, to elucidate the functional significance of splice variants and to investigate mechanisms underlying the fidelity of mRNA splicing, we used an indirect approach based on analyzing the evolutionary conservation of splice variants among species. Using DNA polymerase beta as an indicator locus, we cloned and characterized the types and frequencies of transcripts generated in primary cell lines of five primate species. Overall, we found that in addition to the canonical DNA polymerase beta transcript, there were 25 alternative transcripts generated, most containing premature terminating codons. We used a statistical method borrowed from community ecology to show that there is significant diversity and little conservation in alternative splicing patterns among species, despite high sequence similarity in the underlying genomic (exonic) sequences. However, the frequency of alternative splicing at this locus correlates well with life history parameters such as the maximal longevity of each species, indicating that the alternative splicing of unproductive splice variants may have adaptive significance, even if the specific RNA transcripts themselves have no function. These results demonstrate the validity of the phylogenetic conservation approach in elucidating the biological significance of alternative splicing.

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OB-fold domain of KREPA4 mediates high-affinity interaction with guide RNA and possesses annealing activity.

Publication Date: 2010 Aug 16 PMID: 20713467
Authors: Kala, S. - Salavati, R.
Journal: RNA

KREPA4, also called MP24, is an essential mitochondrial guide RNA (gRNA)-binding protein with a preference for the 3' oligo(U) tail in trypanosomes. Structural prediction and compositional analysis of KREPA4 have identified a conserved OB (oligonucleotide/oligosaccharide-binding)-fold at the C-terminal end and two low compositional complexity regions (LCRs) at its N terminus. Concurrent with these predictions, one or both of these regions in KREPA4 protein may be involved in gRNA binding. To test this possibility, deletion mutants of KREPA4 were made and the effects on the gRNA-binding affinities were measured by quantitative electrophoretic mobility shift assays. The gRNA-binding specificities of these mutants were evaluated by competition experiments using gRNAs with U-tail deletions or stem-loop modifications and uridylated nonguide RNAs or heterologous RNA. Our results identified the predicted OB-fold as the functional domain of KREPA4 that mediates a high-affinity interaction with the gRNA oligo(U) tail. An additional contribution toward RNA-binding function was localized to LCRs that further stabilize the binding through sequence-specific interactions with the guide secondary structure. In this study we also found that the predicted OB-fold has an RNA annealing activity, representing the first report of such activity for a core component of the RNA editing complex.

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Ubiquitous presence of the hammerhead ribozyme motif along the tree of life.

Publication Date: 2010 Aug 12 PMID: 20705646
Authors: de la Pena, M. - Garcia-Robles, I.
Journal: RNA

Examples of small self-cleaving RNAs embedded in noncoding regions already have been found to be involved in the control of gene expression, although their origin remains uncertain. In this work, we show the widespread occurrence of the hammerhead ribozyme (HHR) motif among genomes from the Bacteria, Chromalveolata, Plantae, and Metazoa kingdoms. Intergenic HHRs were detected in three different bacterial genomes, whereas metagenomic data from Galapagos Islands showed the occurrence of similar ribozymes that could be regarded as direct relics from the RNA world. Among eukaryotes, HHRs were detected in the genomes of three water molds as well as 20 plant species, ranging from unicellular algae to vascular plants. These HHRs were very similar to those previously described in small RNA plant pathogens and, in some cases, appeared as close tandem repetitions. A parallel situation of tandemly repeated HHR motifs was also detected in the genomes of lower metazoans from cnidarians to invertebrates, with special emphasis among hematophagous and parasitic organisms. Altogether, these findings unveil the HHR as a widespread motif in DNA genomes, which would be involved in new forms of retrotransposable elements.

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Kinetic and functional analysis of the small RNA methyltransferase HEN1: The catalytic domain is essential for preferential modification of duplex RNA.

Publication Date: 2010 Aug 12 PMID: 20705645
Authors: Vilkaitis, G. - Plotnikova, A. - Klimasauskas, S.
Journal: RNA

The HEN1 RNA methyltransferase from Arabidopsis thaliana catalyzes S-adenosyl-L-methionine (AdoMet)-dependent 2'-O-methylation at the 3'-termini of small double-stranded RNAs and is a crucial factor in the biogenesis of plant small noncoding RNAs, such as miRNAs or siRNAs. We performed functional and kinetic studies of the full-length HEN1 methyltransferase and its truncated form comprising the C-terminal part of the protein (residues 666-942) with a variety of model RNA substrates. Kinetic parameters obtained with natural RNA substrates indicate that HEN1 is highly catalytically efficient in the absence of any supplementary proteins. We find that the enzyme modifies individual strands in succession leading to complete methylation of an RNA duplex. The rates of methyl group transfer to individual strands of hemimethylated substrates under single turnover conditions are comparable with the multiple turnover rate under steady-state conditions, suggesting that release of reaction products is not a rate-limiting event in the reaction cycle. The truncated protein, which includes conserved motifs characteristic for AdoMet binding, efficiently modifies double-stranded RNA substrates in vitro; however, in contrast to the full-length methyltransferase, it shows weaker interactions with both substrates and is sensitive to base mispairing in the first and second positions of the RNA duplex. Our findings suggest an important role for the N-terminal domains in stabilizing the catalytic complex and indicate that major structural determinants required for selective recognition and methylation of RNA duplexes reside in the C-terminal domain.

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Missense suppressor mutations in 16S rRNA reveal the importance of helices h8 and h14 in aminoacyl-tRNA selection.

Publication Date: 2010 Aug 23 PMID: 20699303
Authors: McClory, S. P. - Leisring, J. M. - Qin, D. - Fredrick, K.
Journal: RNA

The molecular basis of the induced-fit mechanism that determines the fidelity of protein synthesis remains unclear. Here, we isolated mutations in 16S rRNA that increase the rate of miscoding and stop codon read-through. Many of the mutations clustered along interfaces between the 30S shoulder domain and other parts of the ribosome, strongly implicating shoulder movement in the induced-fit mechanism of decoding. The largest subset of mutations mapped to helices h8 and h14. These helices interact with each other and with the 50S subunit to form bridge B8. Previous cryo-EM studies revealed a contact between h14 and the switch 1 motif of EF-Tu, raising the possibility that h14 plays a direct role in GTPase activation. To investigate this possibility, we constructed both deletions and insertions in h14. While ribosomes harboring a 2-base-pair (bp) insertion in h14 were completely inactive in vivo, those containing a 2-bp deletion retained activity but were error prone. In vitro, the truncation of h14 accelerated GTP hydrolysis for EF-Tu bearing near-cognate aminoacyl-tRNA, an effect that can largely account for the observed miscoding in vivo. These data show that h14 does not help activate EF-Tu but instead negatively controls GTP hydrolysis by the factor. We propose that bridge B8 normally acts to counter inward rotation of the shoulder domain; hence, mutations in h8 and h14 that compromise this bridge decrease the stringency of aminoacyl-tRNA selection.

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Nucleotides that are essential but not conserved; a sufficient L-tryptophan site in RNA.

Publication Date: 2010 Aug 10 PMID: 20699302
Authors: Majerfeld, I. - Chocholousova, J. - Malaiya, V. - Widmann, J. - McDonald, D. - Reeder, J. - Iyer, M. - Illangasekare, M. - Yarus, M. - Knight, R.
Journal: RNA

Conservation is often used to define essential sequences within RNA sites. However, conservation finds only invariant sequence elements that are necessary for function, rather than finding a set of sequence elements sufficient for function. Biochemical studies in several systems-including the hammerhead ribozyme and the purine riboswitch-find additional elements, such as loop-loop interactions, required for function yet not phylogenetically conserved. Here we define a critical test of sufficiency: We embed a minimal, apparently sufficient motif for binding the amino acid tryptophan in a random-sequence background and ask whether we obtain functional molecules. After a negative result, we use a combination of three-dimensional structural modeling, selection, designed mutations, high-throughput sequencing, and bioinformatics to explore functional insufficiency. This reveals an essential unpaired G in a diverse structural context, varied sequence, and flexible distance from the invariant internal loop binding site identified previously. Addition of the new element yields a sufficient binding site by the insertion criterion, binding tryptophan in 22 out of 23 tries. Random insertion testing for site sufficiency seems likely to be broadly revealing.

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ProbKnot: Fast prediction of RNA secondary structure including pseudoknots.

Publication Date: 2010 Aug 10 PMID: 20699301
Authors: Bellaousov, S. - Mathews, D. H.
Journal: RNA

It is a significant challenge to predict RNA secondary structures including pseudoknots. Here, a new algorithm capable of predicting pseudoknots of any topology, ProbKnot, is reported. ProbKnot assembles maximum expected accuracy structures from computed base-pairing probabilities in O(N(2)) time, where N is the length of the sequence. The performance of ProbKnot was measured by comparing predicted structures with known structures for a large database of RNA sequences with fewer than 700 nucleotides. The percentage of known pairs correctly predicted was 69.3%. Additionally, the percentage of predicted pairs in the known structure was 61.3%. This performance is the highest of four tested algorithms that are capable of pseudoknot prediction. The program is available for download at: http://rna.urmc.rochester.edu/RNAstructure.html.

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Three distinct peptides from the N domain of translation termination factor eRF1 surround stop codon in the ribosome.

Publication Date: 2010 Aug 5 PMID: 20688868
Authors: Bulygin, K. N. - Khairulina, Y. S. - Kolosov, P. M. - Ven'yaminova, A. G. - Graifer, D. M. - Vorobjev, Y. N. - Frolova, L. Y. - Kisselev, L. L. - Karpova, G. G.
Journal: RNA

To study positioning of the polypeptide release factor eRF1 toward a stop signal in the ribosomal decoding site, we applied photoactivatable mRNA analogs, derivatives of oligoribonucleotides. The human eRF1 peptides cross-linked to these short mRNAs were identified. Cross-linkers on the guanines at the second, third, and fourth stop signal positions modified fragment 31-33, and to lesser extent amino acids within region 121-131 (the "YxCxxxF loop") in the N domain. Hence, both regions are involved in the recognition of the purines. A cross-linker at the first uridine of the stop codon modifies Val66 near the NIKS loop (positions 61-64), and this region is important for recognition of the first uridine of stop codons. Since the N domain distinct regions of eRF1 are involved in a stop-codon decoding, the eRF1 decoding site is discontinuous and is not of "protein anticodon" type. By molecular modeling, the eRF1 molecule can be fitted to the A site proximal to the P-site-bound tRNA and to a stop codon in mRNA via a large conformational change to one of its three domains. In the simulated eRF1 conformation, the YxCxxxF motif and positions 31-33 are very close to a stop codon, which becomes also proximal to several parts of the C domain. Thus, in the A-site-bound state, the eRF1 conformation significantly differs from those in crystals and solution. The model suggested for eRF1 conformation in the ribosomal A site and cross-linking data are compatible.

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Demonstrating polymorphic miRNA-mediated gene regulation in vivo: application to the g+6223G->A mutation of Texel sheep.

Publication Date: 2010 Sep PMID: 20679369
Authors: Takeda, H. - Charlier, C. - Farnir, F. - Georges, M.
Journal: RNA

We herein describe the development of a biochemical method to evaluate the effect of single nucleotide polymorphisms (SNPs) in target genes on their regulation by microRNAs in vivo. The method is based on the detection of allelic imbalance in RNAs coimmunoprecipitated with AGO proteins from tissues of heterozygous individuals. We characterize the performances of our approach using a model system in a cell culture, and then apply it successfully to prove that the 3'UTR g+6223G-->A mutation operates by promoting RISC-dependent down-regulation of myostatin (MSTN) in skeletal muscle of Texel sheep.

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Novel endoribonucleases as central players in various pathways of eukaryotic RNA metabolism.

Publication Date: 2010 Sep PMID: 20675404
Authors: Tomecki, R. - Dziembowski, A.
Journal: RNA

For a long time it has been assumed that the decay of RNA in eukaryotes is mainly carried out by exoribonucleases, which is in contrast to bacteria, where endoribonucleases are well documented to initiate RNA degradation. In recent years, several as yet unknown endonucleases have been described, which has changed our view on eukaryotic RNA metabolism. Most importantly, it was shown that the primary eukaryotic 3' --> 5' exonuclease, the exosome complex has the ability to endonucleolytically cleave its physiological RNA substrates, and novel endonucleases involved in both nuclear and cytoplasmic RNA surveillance pathways were discovered concurrently. In addition, endoribonucleases responsible for long-known processing steps in the maturation pathways of various RNA classes were recently identified. Moreover, one of the most intensely studied RNA decay pathways--RNAi--is controlled and stimulated by the action of different endonucleases. Furthermore, endoribonucleolytic cleavages executed by various enzymes are also the hallmark of RNA degradation and processing in plant chloroplasts. Finally, multiple context-specific endoribonucleases control qualitative and/or quantitative changes of selected transcripts under particular conditions in different eukaryotic organisms. The aim of this review is to discuss the impact of all of these discoveries on our current understanding of eukaryotic RNA metabolism.

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Translational competence of ribosomes released from a premature termination codon is modulated by NMD factors.

Publication Date: 2010 Sep PMID: 20675403
Authors: Ghosh, S. - Ganesan, R. - Amrani, N. - Jacobson, A.
Journal: RNA

In addition to their well-documented roles in the promotion of nonsense-mediated mRNA decay (NMD), yeast Upf proteins (Upf1, Upf2/Nmd2, and Upf3) also manifest translational regulatory functions, at least in vitro, including roles in premature translation termination and subsequent reinitiation. Here, we find that all upf Delta strains also fail to reinitiate translation after encountering a premature termination codon (PTC) in vivo, a result that led us to seek a unifying mechanism for all of these translation phenomena. Comparisons of the in vitro translational activities of wild-type (WT) and upf1 Delta extracts were utilized to test for a Upf1 role in post-termination ribosome reutilization. Relative to WT extracts, non-nucleased extracts lacking Upf1 had approximately twofold decreased activity for the translation of synthetic CAN1/LUC mRNA, a defect paralleled by fewer ribosomes per mRNA and reduced efficiency of the 60S joining step at initiation. These deficiencies could be complemented by purified FLAG-Upf1, or 60S subunits, and appeared to reflect diminished cycling of ribosomes from endogenous PTC-containing mRNAs to exogenously added synthetic mRNA in the same extracts. This hypothesis was tested, and supported, by experiments in which nucleased WT or upf1 Delta extracts were first challenged with high concentrations of synthetic mRNAs that were templates for either normal or premature translation termination and then assayed for their capacity to translate a normal mRNA. Our results indicate that Upf1 plays a key role in a mechanism coupling termination and ribosome release at a PTC to subsequent ribosome reutilization for another round of translation initiation.

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Mutations at the accommodation gate of the ribosome impair RF2-dependent translation termination.

Publication Date: 2010 Sep PMID: 20668033
Authors: Burakovsky, D. E. - Sergiev, P. V. - Steblyanko, M. A. - Kubarenko, A. V. - Konevega, A. L. - Bogdanov, A. A. - Rodnina, M. V. - Dontsova, O. A.
Journal: RNA

During protein synthesis, aminoacyl-tRNA (aa-tRNA) and release factors 1 and 2 (RF1 and RF2) have to bind at the catalytic center of the ribosome on the 50S subunit where they take part in peptide bond formation or peptidyl-tRNA hydrolysis, respectively. Computer simulations of aa-tRNA movement into the catalytic site (accommodation) suggested that three nucleotides of 23S rRNA, U2492, C2556, and C2573, form a "gate" at which aa-tRNA movement into the A site is retarded. Here we examined the role of nucleotides C2573 of 23S rRNA, a part of the putative accommodation gate, and of the neighboring A2572 for aa-tRNA binding followed by peptide bond formation and for the RF2-dependent peptide release. Mutations at the two positions did not affect aa-tRNA accommodation, peptide bond formation, or the fidelity of aa-tRNA selection, but impaired RF2-catalyzed peptide release. The data suggest that the ribosome is a robust machine that allows rapid aa-tRNA accommodation despite the defects at the accommodation gate. In comparison, peptide release by RF2 appears more sensitive to these mutations, due to slower accommodation of the factor or effects on RF2 positioning in the A site.

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Characterization of EMU, the Arabidopsis homolog of the yeast THO complex member HPR1.

Publication Date: 2010 Sep PMID: 20668032
Authors: Furumizu, C. - Tsukaya, H. - Komeda, Y.
Journal: RNA

Diverse and precise control is essential for eukaryotic gene expression. This is accomplished through the recruitment of a myriad of proteins to a nascent messenger RNA (mRNA) to mediate modifications, such as capping, splicing, 3'-end processing, and export. Despite being important for every cell, however, the mechanism by which the formation of diverse messenger ribonucleoprotein (mRNP) particles contributes to maintaining intricate systems in the multicellular organism remains incompletely defined. We identified and characterized a mutant gene named erecta mRNA under-expressed (emu) that leads to the defective mRNA accumulation of ERECTA, a developmental regulator in the model plant Arabidopsis thaliana. EMU encodes a protein homologous to a component of the THO complex that is required for the generation of functional mRNPs. Further analysis suggested that EMU is genetically associated with SERRATE, HYPONASTIC LEAVES1, and ARGONAUTE1, which are required for proper RNA maturation or action. Furthermore, mutations in another THO-related gene led to embryonic lethality. These findings support the presence and importance of the THO-related complex in plants as well as yeast and vertebrates.

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Structural and biochemical characterization of CRN-5 and Rrp46: an exosome component participating in apoptotic DNA degradation.

Publication Date: 2010 Sep PMID: 20660080
Authors: Yang, C. C. - Wang, Y. T. - Hsiao, Y. Y. - Doudeva, L. G. - Kuo, P. H. - Chow, S. Y. - Yuan, H. S.
Journal: RNA

Rrp46 was first identified as a protein component of the eukaryotic exosome, a protein complex involved in 3' processing of RNA during RNA turnover and surveillance. The Rrp46 homolog, CRN-5, was subsequently characterized as a cell death-related nuclease, participating in DNA fragmentation during apoptosis in Caenorhabditis elegans. Here we report the crystal structures of CRN-5 and rice Rrp46 (oRrp46) at a resolution of 3.9 A and 2.0 A, respectively. We found that recombinant human Rrp46 (hRrp46), oRrp46, and CRN-5 are homodimers, and that endogenous hRrp46 and oRrp46 also form homodimers in a cellular environment, in addition to their association with a protein complex. Dimeric oRrp46 had both phosphorolytic RNase and hydrolytic DNase activities, whereas hRrp46 and CRN-5 bound to DNA without detectable nuclease activity. Site-directed mutagenesis in oRrp46 abolished either its DNase (E160Q) or RNase (K75E/Q76E) activities, confirming the critical importance of these residues in catalysis or substrate binding. Moreover, CRN-5 directly interacted with the apoptotic nuclease CRN-4 and enhanced the DNase activity of CRN-4, suggesting that CRN-5 cooperates with CRN-4 in apoptotic DNA degradation. Taken together all these results strongly suggest that Rrp46 forms a homodimer separately from exosome complexes and, depending on species, is either a structural or catalytic component of the machinery that cleaves DNA during apoptosis.

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Thermodynamics of RNA melting, one base pair at a time.

Publication Date: 2010 Sep PMID: 20660079
Authors: Nikolova, E. N. - Al-Hashimi, H. M.
Journal: RNA

The melting of base pairs is a ubiquitous feature of RNA structural transitions, which are widely used to sense and respond to cellular stimuli. A recent study employing solution nuclear magnetic resonance (NMR) imino proton exchange spectroscopy provides a rare base-pair-specific view of duplex melting in the Salmonella FourU RNA thermosensor, which regulates gene expression in response to changes in temperature at the translational level by undergoing a melting transition. The authors observe "microscopic" enthalpy-entropy compensation--often seen "macroscopically" across a series of related molecular species--across base pairs within the same RNA. This yields variations in base-pair stabilities that are an order of magnitude smaller than corresponding variations in enthalpy and entropy. A surprising yet convincing link is established between the slopes of enthalpy-entropy correlations and RNA melting points determined by circular dichroism (CD), which argues that unfolding occurs when base-pair stabilities are equalized. A single AG-to-CG mutation, which enhances the macroscopic hairpin thermostability and folding cooperativity and renders the RNA thermometer inactive in vivo, spreads its effect microscopically throughout all base pairs in the RNA, including ones far removed from the site of mutation. The authors suggest that an extended network of hydration underlies this long-range communication. This study suggests that the deconstruction of macroscopic RNA unfolding in terms of microscopic unfolding events will require careful consideration of water interactions.

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A group II intron encodes a functional LAGLIDADG homing endonuclease and self-splices under moderate temperature and ionic conditions.

Publication Date: 2010 Sep PMID: 20656798
Authors: Mullineux, S. T. - Costa, M. - Bassi, G. S. - Michel, F. - Hausner, G.
Journal: RNA

A group II intron encoding a protein belonging to the LAGLIDADG family of homing endonucleases was identified in the mitochondrial rns gene of the filamentous fungus Leptographium truncatum, and the catalytic activities of both the intron and its encoded protein were characterized. A model of the RNA secondary structure indicates that the intron is a member of the IIB1 subclass and the open reading frame is inserted in ribozyme domain III. In vitro assays carried out with two versions of the intron, one in which the open reading frame was removed and the other in which it was present, demonstrate that both versions of the intron readily self-splice at 37 degrees C and at a concentration of MgCl(2) as low as 6 mM. The open reading frame encodes a functional LAGLIDADG homing endonuclease that cleaves 2 (top strand) and 6 (bottom strand) nucleotides (nt) upstream of the intron insertion site, generating 4 nt 3' OH overhangs. In vitro splicing assays carried out in the absence and presence of the intron-encoded protein indicate that the protein does not enhance intron splicing, and RNA-binding assays show that the protein does not appear to bind to the intron RNA precursor transcript. These findings raise intriguing questions concerning the functional and evolutionary relationships of the two components of this unique composite element.

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The roles of phospholipase C activation and alternative ADAR1 and ADAR2 pre-mRNA splicing in modulating serotonin 2C-receptor editing in vivo.

Publication Date: 2010 Sep PMID: 20651031
Authors: Schmauss, C. - Zimnisky, R. - Mehta, M. - Shapiro, L. P.
Journal: RNA

The serotonin 2C receptor (5-HT2CR), a Gq-protein-coupled neurotransmitter receptor, exists in multiple isoforms that result from RNA editing of five exonic adenosines that are converted to inosines. In the adult brain, editing of 5-HT2C pre-mRNA exhibits remarkable plasticity in response to environmental and neurochemical stimuli. Here, we investigated two potential mechanisms underlying these plastic changes in adult 5-HT2CR editing phenotypes in vivo: activation of phospholipase C (PLC) and alternative splicing of pre-mRNA encoding the editing enzymes ADAR1 and ADAR2. Studies on two inbred strains of mice (C57Bl/6 and Balb/c) revealed that sustained stimulation of PLC--a downstream effector of activated G alpha q protein--increased editing of forebrain neocortical 5-HT2C pre-mRNA at two sites known to be targeted by ADAR2. Moreover, changes in relative expression of the alternatively spliced "a" and "b" mRNA isoforms of ADAR1 and ADAR2 also correlate with changes in 5-HT2CR editing. The site-specific changes in 5-HT2CR editing detected in mice with different "a" over "b" ADAR mRNA isoform ratios only partially overlap with those evoked by sustained PLC activation and are best explained by the increased editing efficiency of ADAR1. Thus, activation of PLC and alternative splicing of ADAR pre-mRNA have both overlapping and specific roles in modulating 5-HT2CR editing phenotypes.

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A comprehensive analysis of translational missense errors in the yeast Saccharomyces cerevisiae.

Publication Date: 2010 Sep PMID: 20651030
Authors: Kramer, E. B. - Vallabhaneni, H. - Mayer, L. M. - Farabaugh, P. J.
Journal: RNA

The process of protein synthesis must be sufficiently rapid and sufficiently accurate to support continued cellular growth. Failure in speed or accuracy can have dire consequences, including disease in humans. Most estimates of the accuracy come from studies of bacterial systems, principally Escherichia coli, and have involved incomplete analysis of possible errors. We recently used a highly quantitative system to measure the frequency of all types of misreading errors by a single tRNA in E. coli. That study found a wide variation in error frequencies among codons; a major factor causing that variation is competition between the correct (cognate) and incorrect (near-cognate) aminoacyl-tRNAs for the mutant codon. Here we extend that analysis to measure the frequency of missense errors by two tRNAs in a eukaryote, the yeast Saccharomyces cerevisiae. The data show that in yeast errors vary by codon from a low of 4 x 10(-5) to a high of 6.9 x 10(-4) per codon and that error frequency is in general about threefold lower than in E. coli, which may suggest that yeast has additional mechanisms that reduce missense errors. Error rate again is strongly influenced by tRNA competition. Surprisingly, missense errors involving wobble position mispairing were much less frequent in S. cerevisiae than in E. coli. Furthermore, the error-inducing aminoglycoside antibiotic, paromomycin, which stimulates errors on all error-prone codons in E. coli, has a more codon-specific effect in yeast.

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Functional implications of the emergence of alternative splicing in hnRNP A/B transcripts.

Publication Date: 2010 Sep PMID: 20651029
Authors: Han, S. P. - Kassahn, K. S. - Skarshewski, A. - Ragan, M. A. - Rothnagel, J. A. - Smith, R.
Journal: RNA

The heterogeneous nuclear ribonucleoproteins (hnRNPs) A/B are a family of RNA-binding proteins that participate in various aspects of nucleic acid metabolism, including mRNA trafficking, telomere maintenance, and splicing. They are both regulators and targets of alternative splicing, and the patterns of alternative splicing of their transcripts have diverged between paralogs and between orthologs in different species. Surprisingly, the extent of this splicing variation and its implications for post-transcriptional regulation have remained largely unexplored. Here, we conducted a detailed analysis of hnRNP A/B sequences and expression patterns across six vertebrates. Alternative exons emerged via the introduction of new splice sites, changes in the strengths of existing splice sites, and the accumulation of auxiliary splicing regulatory motifs. Observed isoform expression patterns could be attributed to the frequency and strength of cis-elements. We found a trend toward increased splicing variation in mammals and identified novel alternatively spliced isoforms in human and chicken. Pulldown and translational assays demonstrated that the inclusion of alternative exons altered the affinity of hnRNP A/B proteins for their cognate nucleic acids and modified protein expression levels. As the hnRNPs A/B regulate several key steps in mRNA processing, the involvement of diverse hnRNP isoforms in multiple cellular contexts and species implies concomitant differences in the transcriptional output of these systems. We conclude that the emergence of alternative splicing in the hnRNPs A/B has contributed to the diversification of their roles in the regulation of alternative splicing and has thus added an unexpected layer of regulatory complexity to transcription in vertebrates.

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Turning limited experimental information into 3D models of RNA.

Publication Date: 2010 Sep PMID: 20651028
Authors: Flores, S. C. - Altman, R. B.
Journal: RNA

Our understanding of RNA functions in the cell is evolving rapidly. As for proteins, the detailed three-dimensional (3D) structure of RNA is often key to understanding its function. Although crystallography and nuclear magnetic resonance (NMR) can determine the atomic coordinates of some RNA structures, many 3D structures present technical challenges that make these methods difficult to apply. The great flexibility of RNA, its charged backbone, dearth of specific surface features, and propensity for kinetic traps all conspire with its long folding time, to challenge in silico methods for physics-based folding. On the other hand, base-pairing interactions (either in runs to form helices or isolated tertiary contacts) and motifs are often available from relatively low-cost experiments or informatics analyses. We present RNABuilder, a novel code that uses internal coordinate mechanics to satisfy user-specified base pairing and steric forces under chemical constraints. The code recapitulates the topology and characteristic L-shape of tRNA and obtains an accurate noncrystallographic structure of the Tetrahymena ribozyme P4/P6 domain. The algorithm scales nearly linearly with molecule size, opening the door to the modeling of significantly larger structures.

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Of proteins and RNA: the RNase P/MRP family.

Publication Date: 2010 Sep PMID: 20627997
Authors: Esakova, O. - Krasilnikov, A. S.
Journal: RNA

Nuclear ribonuclease (RNase) P is a ubiquitous essential ribonucleoprotein complex, one of only two known RNA-based enzymes found in all three domains of life. The RNA component is the catalytic moiety of RNases P across all phylogenetic domains; it contains a well-conserved core, whereas peripheral structural elements are diverse. RNA components of eukaryotic RNases P tend to be less complex than their bacterial counterparts, a simplification that is accompanied by a dramatic reduction of their catalytic ability in the absence of protein. The size and complexity of the protein moieties increase dramatically from bacterial to archaeal to eukaryotic enzymes, apparently reflecting the delegation of some structural functions from RNA to proteins and, perhaps, in response to the increased complexity of the cellular environment in the more evolutionarily advanced organisms; the reasons for the increased dependence on proteins are not clear. We review current information on RNase P and the closely related universal eukaryotic enzyme RNase MRP, focusing on their functions and structural organization.

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Retention of spliceosomal components along ligated exons ensures efficient removal of multiple introns.

Publication Date: 2010 Sep PMID: 20610656
Authors: Crabb, T. L. - Lam, B. J. - Hertel, K. J.
Journal: RNA

The majority of mammalian pre-mRNAs contains multiple introns that are excised prior to export and translation. After intron excision, ligated exon intermediates participate in subsequent intron excisions. However, exon ligation generates an exon of increased size, a feature of pre-mRNA splicing that can interfere with downstream splicing events. These considerations raise the question of whether unique mechanisms exist that permit efficient removal of introns neighboring ligated exons. Kinetic analyses of multiple intron-containing pre-mRNAs revealed that splicing is more efficient following an initial intron removal event, suggesting that either the recruitment of the exon junction complex (EJC) to ligated exons increases the efficiency of multiple intron excisions or that the initial definition of splice sites is sufficient to permit efficient splicing of introns neighboring ligated exons. Knockdown experiments show that the deposition of the EJC does not affect subsequent splicing kinetics. Instead, spliceosomal components that are not involved in the initial splicing event remain associated with the pre-mRNA to ensure efficient removal of neighboring introns. Thus, ligated exons do not require redefinition, providing an additional kinetic advantage for exon defined splice sites.

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