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NAR Top Articles - RNA


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April 2015

Translation rate is controlled by coupled trade-offs between site accessibility, selective RNA unfolding and sliding at upstream standby sites
Borujeni, AE; Channarasappa, AS; Salis, HM
Nucleic Acids Res. 2014, 42, 2646-2659
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The ribosome's interactions with mRNA govern its translation rate and the effects of post-transcriptional regulation. Long, structured 5' untranslated regions (5' UTRs) are commonly found in bacterial mRNAs, though the physical mechanisms that determine how the ribosome binds these upstream regions remain poorly defined. Here, we systematically investigate the ribosome's interactions with structured standby sites, upstream of Shine-Dalgarno sequences, and show that these interactions can modulate translation initiation rates by over 100-fold. We find that an mRNA's translation initiation rate is controlled by the amount of single-stranded surface area, the partial unfolding of RNA structures to minimize the ribosome's binding free energy penalty, the absence of cooperative binding and the potential for ribosomal sliding. We develop a biophysical model employing thermodynamic first principles and a four-parameter free energy model to accurately predict the ribosome's translation initiation rates for 136 synthetic 5' UTRs with large structures, diverse shapes and multiple standby site modules...

Exosomes in human semen carry a distinctive repertoire of small non-coding RNAs with potential regulatory functions
Vojtech, L; Woo, S; Hughes, S; Levy, C; Ballweber, L; Sauteraud, RP; Strobl, J; Westerberg, K; Gottardo, R; Tewari, M; Hladik, F
Nucleic Acids Res. 2014, 42, 7290-7304
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Semen contains relatively ill-defined regulatory components that likely aid fertilization, but which could also interfere with defense against infection. Each ejaculate contains trillions of exosomes, membrane-enclosed subcellular microvesicles, which have immunosuppressive effects on cells important in the genital mucosa. Exosomes in general are believed to mediate inter-cellular communication, possibly by transferring small RNA molecules. We found that seminal exosome (SE) preparations contain a substantial amount of RNA from 20 to 100 nucleotides (nts) in length. We sequenced 20-40 and 40-100 nt fractions of SE RNA separately from six semen donors. We found various classes of small non-coding RNA, including microRNA (21.7% of the RNA in the 20-40 nt fraction) as well as abundant Y RNAs and tRNAs present in both fractions. Specific RNAs were consistently present in all donors. For example, 10 (of similar to 2600 known) microRNAs constituted over 40% of mature microRNA in SE. Additionally, tRNA fragments were strongly enriched for 5'-ends of 18-19 or 30-34 nts in length...

All three RNA recognition motifs and the hinge region of HuC play distinct roles in the regulation of alternative splicing
Hinman, MN; Zhou, HL; Sharma, A; Lou, H
Nucleic Acids Res. 2013, 41, 5049-5061
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The four Hu [embryonic lethal abnormal vision-like (ELAVL)] protein family members regulate alternative splicing by binding to U-rich sequences surrounding target exons and affecting the interaction of the splicing machinery and/or local chromatin modifications. Each of the Hu proteins contains a divergent N-terminus, three highly conserved RNA recognition motifs (RRM1, RRM2 and RRM3) and a hinge region separating RRM2 and RRM3. The roles of each domain in splicing regulation are not well understood. Here, we investigate how HuC, a relatively poorly characterized family member, regulates three target pre-mRNAs: neurofibromatosis type I, Fas and HuD. We find that the HuC N-terminus is dispensable for splicing regulation, and the three RRMs are required for splicing regulation of each target, whereas the hinge region contributes to regulation of only some targets. Interestingly, the regions of the hinge and RRM3 required for regulating different targets only partially overlap, implying substrate-specific mechanisms of HuC-mediated splicing regulation. We show that RRM1 and RRM2 are required for binding to target pre-mRNAs...

5'' isomiR variation is of functional and evolutionary importance
Tan, GC; Chan, E; Molnar, A; Sarkar, R; Alexieva, D; Isa, IM; Robinson, S; Zhang, SC; Ellis, P; Langford, CF; Guillot, PV; Chandrashekran, A; Fisk, NM; Castellano, L; Meister, G; Winston, RM; Cui, W; Baulcombe, D; Dibb, NJ
Nucleic Acids Res. 2014, 42, 9424-9435
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We have sequenced miRNA libraries from human embryonic, neural and foetal mesenchymal stem cells. We report that the majority of miRNA genes encode mature isomers that vary in size by one or more bases at the 3' and/or 5' end of the miRNA. Northern blotting for individual miRNAs showed that the proportions of isomiRs expressed by a single miRNA gene often differ between cell and tissue types. IsomiRs were readily co-immunoprecipitated with Argonaute proteins in vivo and were active in luciferase assays, indicating that they are functional. Bioinformatics analysis predicts substantial differences in targeting between miRNAs with minor 5' differences and in support of this we report that a 5' isomiR-9-1 gained the ability to inhibit the expression of DNMT3B and NCAM2 but lost the ability to inhibit CDH1 in vitro. This result was confirmed by the use of isomiR-specific sponges. Our analysis of the miRGator database indicates that a small percentage of human miRNA genes express isomiRs as the dominant transcript in certain cell types...

Cytoplasmic and nuclear quality control and turnover of single-stranded RNA modulate post-transcriptional gene silencing in plants
Moreno, AB; de Alba, AEM; Bardou, F; Crespi, MD; Vaucheret, H; Maizel, A; Mallory, AC
Nucleic Acids Res. 2013, 41, 4699-4708
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Eukaryotic RNA quality control (RQC) uses both endonucleolytic and exonucleolytic degradation to eliminate dysfunctional RNAs. In addition, endogenous and exogenous RNAs are degraded through post-transcriptional gene silencing (PTGS), which is triggered by the production of double-stranded (ds)RNAs and proceeds through short-interfering (si)RNA-directed ARGONAUTE-mediated endonucleolytic cleavage. Compromising cytoplasmic or nuclear 5'-3' exoribonuclease function enhances sense-transgene (S)-PTGS in Arabidopsis, suggesting that these pathways compete for similar RNA substrates. Here, we show that impairing nonsense-mediated decay, deadenylation or exosome activity enhanced S-PTGS, which requires host RNA-dependent RNA polymerase 6 (RDR6/SGS2/SDE1) and SUPPRESSOR OF GENE SILENCING 3 (SGS3) for the transformation of single-stranded RNA into dsRNA to trigger PTGS. However, these RQC mutations had no effect on inverted-repeat-PTGS, which directly produces hairpin dsRNA through transcription. Moreover, we show that these RQC factors are nuclear and cytoplasmic and are found in two RNA degradation foci in the cytoplasm...

A dynamic alternative splicing program regulates gene expression during terminal erythropoiesis
Pimentel, H; Parra, M; Gee, S; Ghanem, D; An, XL; Li, J; Mohandas, N; Pachter, L; Conboy, JG
Nucleic Acids Res. 2014, 42, 4031-4042
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Alternative pre-messenger RNA splicing remodels the human transcriptome in a spatiotemporal manner during normal development and differentiation. Here we explored the landscape of transcript diversity in the erythroid lineage by RNA-seq analysis of five highly purified populations of morphologically distinct human erythroblasts, representing the last four cell divisions before enucleation. In this unique differentiation system, we found evidence of an extensive and dynamic alternative splicing program encompassing genes with many diverse functions. Alternative splicing was particularly enriched in genes controlling cell cycle, organelle organization, chromatin function and RNA processing. Many alternative exons exhibited differentiation-associated switches in splicing efficiency, mostly in late-stage polychromatophilic and orthochromatophilic erythroblasts, in concert with extensive cellular remodeling that precedes enucleation. A subset of alternative splicing switches introduces premature translation termination codons into selected transcripts in a differentiation stage-specific manner...

Quantifying sequence and structural features of protein-RNA interactions
Li, SL; Yamashita, K; Amada, KM; Standley, DM
Nucleic Acids Res. 2014, 42, 10086-10098
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Increasing awareness of the importance of protein-RNA interactions has motivated many approaches to predict residue-level RNA binding sites in proteins based on sequence or structural characteristics. Sequence-based predictors are usually high in sensitivity but low in specificity; conversely structure-based predictors tend to have high specificity, but lower sensitivity. Here we quantified the contribution of both sequence-and structure-based features as indicators of RNA-binding propensity using a machine-learning approach. In order to capture structural information for proteins without a known structure, we used homology modeling to extract the relevant structural features. Several novel and modified features enhanced the accuracy of residue-level RNA-binding propensity beyond what has been reported previously, including by meta-prediction servers. These features include: hidden Markov model-based evolutionary conservation, surface deformations based on the Laplacian norm formalism, and relative solvent accessibility partitioned into backbone and side chain contributions. We constructed a web server called aaRNA that implements the proposed method and demonstrate its use in identifying putative RNA binding site

7SL RNA represses p53 translation by competing with HuR
Abdelmohsen, K; Panda, AC; Kang, MJ; Guo, R; Kim, J; Grammatikakis, I; Yoon, JH; Dudekula, DB; Noh, JH; Yang, XL; Martindale, JL; Gorospe, M
Nucleic Acids Res. 2014, 42, 10099-10111
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Noncoding RNAs (ncRNAs) and RNA-binding proteins are potent post-transcriptional regulators of gene expression. The ncRNA 7SL is upregulated in cancer cells, but its impact upon the phenotype of cancer cells is unknown. Here, we present evidence that 7SL forms a partial hybrid with the 3' untranslated region (UTR) of TP53 mRNA, which encodes the tumor suppressor p53. The interaction of 7SL with TP53 mRNA reduced p53 translation, as determined by analyzing p53 expression levels, nascent p53 translation and TP53 mRNA association with polysomes. Silencing 7SL led to increased binding of HuR to TP53 mRNA, an interaction that led to the promotion of p53 translation and increased p53 abundance. We propose that the competition between 7SL and HuR for binding to TP53 3' UTR contributes to determining the magnitude of p53 translation, in turn affecting p53 levels and the growth-suppressive function of p53. Our findings suggest that targeting 7SL may be effective in the treatment of cancers with reduced p53 levels.

Interplay between pre-mRNA splicing and microRNA biogenesis within the supraspliceosome
Agranat-Tamir, L; Shomron, N; Sperling, J; Sperling, R
Nucleic Acids Res. 2014, 42, 4640-4651
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MicroRNAs (miRNAs) are central regulators of gene expression, and a large fraction of them are encoded in introns of RNA polymerase II transcripts. Thus, the biogenesis of intronic miRNAs by the microprocessor and the splicing of their host introns by the spliceosome require coordination between these processing events. This cross-talk is addressed here. We show that key microprocessor proteins Drosha and DGCR8 as well as pre-miRNAs cosediment with supraspliceosomes, where nuclear posttranscriptional processing is executed. We further show that inhibition of splicing increases miRNAs expression, whereas knock-down of Drosha increases splicing. We identified a novel splicing event in intron 13 of MCM7, where the miR-106b-25 cluster is located. The unique splice isoform includes a hosted pre-miRNA in the extended exon and excludes its processing. This indicates a possible mechanism of altering the levels of different miRNAs originating from the same transcript. Altogether, our study indicates interplay between the splicing and microprocessor machineries within a supraspliceosome context.

Free mRNA in excess upon polysome dissociation is a scaffold for protein multimerization to form stress granules
Bounedjah, O; Desforges, B; Wu, TD; Pioche-Durieu, C; Marco, S; Hamon, L; Curmi, PA; Guerquin-Kern, JL; Pietrement, O; Pastre, D
Nucleic Acids Res. 2014, 42, 8678-8691
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The sequence of events leading to stress granule assembly in stressed cells remains elusive. We show here, using isotope labeling and ion microprobe, that proportionally more RNA than proteins are present in stress granules than in surrounding cytoplasm. We further demonstrate that the delivery of single strand polynucleotides, mRNA and ssDNA, to the cytoplasm can trigger stress granule assembly. On the other hand, increasing the cytoplasmic level of mRNA-binding proteins like YB-1 can directly prevent the aggregation of mRNA by forming isolated mRNPs, as evidenced by atomic force microscopy. Interestingly, we also discovered that enucleated cells do form stress granules, demonstrating that the translocation to the cytoplasm of nuclear prion-like RNA-binding proteins like TIA-1 is dispensable for stress granule assembly. The results lead to an alternative view on stress granule formation based on the following sequence of events: after the massive dissociation of polysomes during stress, mRNA-stabilizing proteins like YB-1 are outnumbered by the burst of nonpolysomal mRNA...

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