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NAR Top Articles - RNA


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October 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...

A complete landscape of post-transcriptional modifications in mammalian mitochondrial tRNAs
Suzuki, T; Suzuki, T
Nucleic Acids Res. 2014, 42, 7346-7357
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In mammalian mitochondria, 22 species of tRNAs encoded in mitochondrial DNA play crucial roles in the translation of 13 essential subunits of the respiratory chain complexes involved in oxidative phosphorylation. Following transcription, mitochondrial tRNAs are modified by nuclear-encoded tRNA-modifying enzymes. These modifications are required for the proper functioning of mitochondrial tRNAs (mt tRNAs), and the absence of these modifications can cause pathological consequences. To date, however, the information available about these modifications has been incomplete. To address this issue, we isolated all 22 species of mt tRNAs from bovine liver and comprehensively determined the post-transcriptional modifications in each tRNA by mass spectrometry. Here, we describe the primary structures with post-transcriptional modifications of seven species of mt tRNAs which were previously uncharacterized, and provide revised information regarding base modifications in five other mt tRNAs. In the complete set of bovine mt tRNAs, we found 15 species of modified nucleosides at 118 positions (7.48% of total bases). This result provides insight into the molecular mechanisms underlying the decoding system in mammalian mitochondria...

Stability of RNA quadruplex in open reading frame determines proteolysis of human estrogen receptor α
Endoh, T; Kawasaki, Y; Sugimoto, N
Nucleic Acids Res. 2013, 41, 6222-6231
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mRNAs encodes not only information that determines amino acid sequences but also additional layers of information that regulate the translational processes. Notably, translational halt at specific position caused by rare codons or stable RNA structures is one of the potential factors regulating the protein expressions and structures. In this study, a quadruplex-forming potential (QFP) sequence derived from an open reading frame of human estrogen receptor alpha (hER alpha) mRNA was revealed to form parallel G-quadruplex and halt the translation elongation in vitro. Moreover, when the full-length hER alpha and variants containing synonymous mutations in the QFP sequence were expressed in cells, translation products cleaved at specific site were observed in quantities dependent on the thermodynamic stability of the G-quadruplexes. These results suggest that the G-quadruplex formation in the coding region of the hER alpha mRNA impacts folding and proteolysis of hER alpha protein by slowing down or temporarily stalling the translation elongation.

Comparative RNA-Seq based dissection of the regulatory networks and environmental stimuli underlying Vibrio parahaemolyticus gene expression during infection
Livny, J; Zhou, XH; Mandlik, A; Hubbard, T; Davis, BM; Waldor, MK
Nucleic Acids Res. 2014, 42, 12212-12223
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Vibrio parahaemolyticus is the leading worldwide cause of seafood-associated gastroenteritis, yet little is known regarding its intraintestinal gene expression or physiology. To date, in vivo analyses have focused on identification and characterization of virulence factors-e. g. a crucial Type III secretion system (T3SS2)-rather than genome-wide analyses of in vivo biology. Here, we used RNA-Seq to profile V. parahaemolyticus gene expression in infected infant rabbits, which mimic human infection. Comparative transcriptomic analysis of V. parahaemolyticus isolated from rabbit intestines and from several laboratory conditions enabled identification of mRNAs and sRNAs induced during infection and of regulatory factors that likely control them. More than 12% of annotated V. parahaemolyticus genes are differentially expressed in the intestine, including the genes of T3SS2, which are likely induced by bile-mediated activation of the transcription factor VtrB. Our analyses also suggest that V. parahaemolyticus has access to glucose or other preferred carbon sources in vivo, but that iron is inconsistently available.

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...

Is an observed non-co-linear RNA product spliced in trans, in cis or just in vitro?
Yu, CY; Liu, HJ; Hung, LY; Kuo, HC; Chuang, TJ
Nucleic Acids Res. 2014, 42, 9410-9423
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Global transcriptome investigations often result in the detection of an enormous number of transcripts composed of non-co-linear sequence fragments. Such 'aberrant' transcript products may arise from post-transcriptional events or genetic rearrangements, or may otherwise be false positives (sequencing/alignment errors or in vitro artifacts). Moreover, post-transcriptionally non-co-linear ('PtNcl') transcripts can arise from trans-splicing or back-splicing in cis (to generate so-called 'circular RNA'). Here, we collected previously-predicted human non-co-linear RNA candidates, and designed a validation procedure integrating in silico filters with multiple experimental validation steps to examine their authenticity. We showed that >50% of the tested candidates were in vitro artifacts, even though some had been previously validated by RT-PCR. After excluding the possibility of genetic rearrangements, we distinguished between trans-spliced and circular RNAs, and confirmed that these two splicing forms can share the same non-co-linear junction...

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...

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...

Negative regulation of the interferon response by an interferon-induced long non-coding RNA
Kambara, H; Niazi, F; Kostadinova, L; Moonka, DK; Siegel, CT; Post, AB; Carnero, E; Barriocanal, M; Fortes, P; Anthony, DD; Valadkhan, S
Nucleic Acids Res. 2014, 42, 10668-10680
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Long non-coding RNAs (lncRNAs) play critical roles in diverse cellular processes; however, their involvement in many critical aspects of the immune response including the interferon (IFN) response remains poorly understood. To address this gap, we compared the global gene expression pattern of primary human hepatocytes before and at three time points after treatment with IFN-alpha. Among similar to 200 IFN-induced lncRNAs, one transcript showed similar to 100-fold induction. This RNA, which we named lncRNA-CMPK2, was a spliced, polyadenylated nuclear transcript that was induced by IFN in diverse cell types from human and mouse. Similar to protein-coding IFN-stimulated genes (ISGs), its induction was dependent on JAK-STAT signaling. Intriguingly, knockdown of lncRNA-CMPK2 resulted in a marked reduction in HCV replication in IFN-stimulated hepatocytes, suggesting that it could affect the antiviral role of IFN. We could show that lncRNA-CMPK2 knockdown resulted in upregulation of several protein-coding antiviral ISGs...

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