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June 2015


Predominance of spliceosomal complex formation over polyadenylation site selection in TDP-43 autoregulation
Bembich, S; Herzog, JS; De Conti, L; Stuani, C; Avendano-Vazquez, SE; Buratti, E; Baralle, M; Baralle, FE
Nucleic Acids Res. 2014, 42, 3362-3371
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TDP-43 is a nuclear protein involved in many aspects of RNA metabolism. To ensure cellular viability, its expression levels within cells must be tightly regulated. We have previously demonstrated that TDP-43 autoregulation occurs through the activation of a normally silent intron in its 3'-UTR sequence that results in the use of alternative polyadenylation sites. In this work, we analyse which is the dominant event in autoregulation: the recognition of the splice sites of 3'-UTR intron 7 or the intrinsic quality of the alternative polyadenylation sites. A panel of minigene constructs was tested for autoregulation functionality, protein production and subcellular messenger RNA localization. Our data clearly indicate that constitutive spliceosome complex formation across intron 7 does not lead to high protein production but, on the contrary, to lower TDP-43 messenger RNA and protein levels. This is due to altered nucleocytoplasmic distribution of the RNA that is mostly retained in the nucleus and degraded. This study provides a novel in-depth characterization of how RNA binding proteins can autoregulate their own levels...

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...

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...

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...

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...

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.

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...

Phosphorothioate oligonucleotides can displace NEAT1 RNA and form nuclear paraspeckle-like structures
Shen, W; Liang, XH; Crooke, ST
Nucleic Acids Res. 2014, 42, 8648-8662
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Nuclear paraspeckles are built co-transcriptionally around a long non-coding RNA, NEAT1. Here we report that transfected 20-mer phosphorothioate-modified (PS) antisense oligonucleotides (ASOs) can recruit paraspeckle proteins to form morphologically normal and apparently functional paraspeckle-like structures containing no NEAT1 RNA. PS-ASOs can associate with paraspeckle proteins, including P54nrb, PSF, PSPC1 and hnRNPK. NEAT1 RNA can be displaced by transfected PS-ASO from paraspeckles and rapidly degraded. Co-localization of PS-ASOs with P54nrb was observed in canonical NEAT1-containing paraspeckles, in perinucleolar caps upon transcriptional inhibition, and importantly, in paraspeckle-like or filament structures lacking NEAT1 RNA. The induced formation of paraspeckle-like and filament structures occurred in mouse embryonic stem cells expressing little or no NEAT1 RNA, suggesting that PS-ASOs can serve as seeding molecules to assemble paraspeckle-like foci in the absence of NEAT1 RNA. Moreover, CTN, an RNA reported to be functionally retained in paraspeckles, was also observed to localize to paraspeckle-like structures, implying that paraspeckle-like structures assembled on PS-ASOs are functional...

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...

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...

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