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January 2015


Negative regulation of the interferon response by an interferon-induced long non-coding RNA
H. Kambara, F. Niazi, L. Kostadinova, D. K. Moonka, C. T. Siegel, A. B. Post, E. Carnero, M. Barriocanal, P. Fortes, D. D. Anthony and S. Valadkhan
Nucleic Acids Res. (2014) 42 (16): 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 approximately 200 IFN-induced lncRNAs, one transcript showed approximately 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...

Arabidopsis thaliana LSM proteins function in mRNA splicing and degradation
A. Golisz, P. J. Sikorski, K. Kruszka and J. Kufel
Nucleic Acids Res. (2013) 41 (12): 6232-6249
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Sm-like (Lsm) proteins have been identified in all organisms and are related to RNA metabolism. Here, we report that Arabidopsis nuclear AtLSM8 protein, as well as AtLSM5, which localizes to both the cytoplasm and nucleus, function in pre-mRNA splicing, while AtLSM5 and the exclusively cytoplasmic AtLSM1 contribute to 5'-3' mRNA decay. In lsm8 and sad1/lsm5 mutants, U6 small nuclear RNA (snRNA) was reduced and unspliced mRNA precursors accumulated, whereas mRNA stability was mainly affected in plants lacking AtLSM1 and AtLSM5. Some of the mRNAs affected in lsm1a lsm1b and sad1/lsm5 plants were also substrates of the cytoplasmic 5'-3' exonuclease AtXRN4 and of the decapping enzyme AtDCP2. Surprisingly, a subset of substrates was also stabilized in the mutant lacking AtLSM8, which supports the notion that plant mRNAs are actively degraded in the nucleus. Localization of LSM components, purification of LSM-interacting proteins as well as functional analyses strongly suggest that at least two LSM complexes with conserved activities in RNA metabolism, AtLSM1-7 and AtLSM2-8, exist also in plants.

Quantifying sequence and structural features of protein-RNA interactions
S. Li, K. Yamashita, K. M. Amada and D. M. Standley
Nucleic Acids Res. (2014) 42 (15): 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...

Exosomes in human semen carry a distinctive repertoire of small non-coding RNAs with potential regulatory functions
L. Vojtech, S. Woo, S. Hughes, C. Levy, L. Ballweber, R. P. Sauteraud, J. Strobl, K. Westerberg, R. Gottardo, M. Tewari and F. Hladik
Nucleic Acids Res. (2014) 42 (11): 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 approximately 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...

A dynamic alternative splicing program regulates gene expression during terminal erythropoiesis
H. Pimentel, M. Parra, S. Gee, D. Ghanem, X. An, J. Li, N. Mohandas, L. Pachter and J. G. Conboy
Nucleic Acids Res. (2014) 42 (6): 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...

Free mRNA in excess upon polysome dissociation is a scaffold for protein multimerization to form stress granules
O. Bounedjah, B. Desforges, T. D. Wu, C. Pioche-Durieu, S. Marco, L. Hamon, P. A. Curmi, J. L. Guerquin-Kern, O. Pietrement and D. Pastre
Nucleic Acids Res. (2014) 42 (13): 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...

HIV-1 Rev protein specifies the viral RNA export pathway by suppressing TAP/NXF1 recruitment
I. Taniguchi, N. Mabuchi and M. Ohno
Nucleic Acids Res. (2014) 42 (10): 6645-6658
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Nuclear RNA export pathways in eukaryotes are often linked to the fate of a given RNA. Therefore, the choice of export pathway should be well-controlled to avoid an unfavorable effect on gene expression. Although some RNAs could be exported by more than one pathway, little is known about how the choice is regulated. This issue is highlighted when the human immunodeficiency virus type 1 (HIV-1) Rev protein induces the export of singly spliced and unspliced HIV-1 transcripts. How these RNAs are exported is not well understood because such transcripts should have the possibility of utilizing CRM1-dependent export via Rev or cellular TAP/NXF1-dependent export via the transcription/export (TREX) complex, or both. Here we found that Rev suppressed TAP/NXF1-dependent export of model RNA substrates that recapitulated viral transcripts. In this effect, Rev interacted with the cap-binding complex and inhibited the recruitment of the TREX complex. Thus, Rev controls the identity of the factor occupying the cap-proximal region that determines the RNA export pathway. This ribonucleoprotein remodeling activity of Rev may favor viral gene expression.

Functional characterization of C. elegans Y-box-binding proteins reveals tissue-specific functions and a critical role in the formation of polysomes
A. Arnold, M. M. Rahman, M. C. Lee, S. Muehlhaeusser, I. Katic, D. Gaidatzis, D. Hess, C. Scheckel, J. E. Wright, A. Stetak, P. R. Boag and R. Ciosk
Nucleic Acids Res. (2014) 42 (21): 13353-13369
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The cold shock domain is one of the most highly conserved motifs between bacteria and higher eukaryotes. Y-box-binding proteins represent a subfamily of cold shock domain proteins with pleiotropic functions, ranging from transcription in the nucleus to translation in the cytoplasm. These proteins have been investigated in all major model organisms except Caenorhabditis elegans. In this study, we set out to fill this gap and present a functional characterization of CEYs, the C. elegans Y-box-binding proteins. We find that, similar to other organisms, CEYs are essential for proper gametogenesis. However, we also report a novel function of these proteins in the formation of large polysomes in the soma. In the absence of the somatic CEYs, polysomes are dramatically reduced with a simultaneous increase in monosomes and disomes, which, unexpectedly, has no obvious impact on animal biology. Because transcripts that are enriched in polysomes in wild-type animals tend to be less abundant in the absence of CEYs...

Interplay between pre-mRNA splicing and microRNA biogenesis within the supraspliceosome
L. Agranat-Tamir, N. Shomron, J. Sperling and R. Sperling
Nucleic Acids Res. (2014) 42 (7): 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.

The protein kinase TOUSLED facilitates RNAi in Arabidopsis
M. N. Uddin, P. Dunoyer, G. Schott, S. Akhter, C. Shi, W. J. Lucas, O. Voinnet and J. Y. Kim
Nucleic Acids Res. (2014) 42 (12): 7971-7980
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RNA silencing is an evolutionarily conserved mechanism triggered by double-stranded RNA that is processed into 21- to 24-nt small interfering (si)RNA or micro (mi)RNA by RNaseIII-like enzymes called Dicers. Gene regulations by RNA silencing have fundamental implications in a large number of biological processes that include antiviral defense, maintenance of genome integrity and the orchestration of cell fates. Although most generic or core components of the various plant small RNA pathways have been likely identified over the past 15 years, factors involved in RNAi regulation through post-translational modifications are just starting to emerge, mostly through forward genetic studies. A genetic screen designed to identify factors required for RNAi in Arabidopsis identified the serine/threonine protein kinase, TOUSLED (TSL). Mutations in TSL affect exogenous and virus-derived siRNA activity in a manner dependent upon its kinase activity. By contrast, despite their pleiotropic developmental phenotype, tsl mutants show no defect in biogenesis or activity of miRNA or endogenous trans-acting siRNA...

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