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August 2015

Trans-splicing and RNA editing of LSU rRNA in Diplonema mitochondria
Valach, M; Moreira, S; Kiethega, GN; Burger, G
Nucleic Acids Res. 2014, 42, 2660-2672
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Mitochondrial ribosomal RNAs (rRNAs) often display reduced size and deviant secondary structure, and sometimes are fragmented, as are their corresponding genes. Here we report a mitochondrial large subunit rRNA (mt-LSU rRNA) with unprecedented features. In the protist Diplonema, the rnl gene is split into two pieces (modules 1 and 2, 534- and 352-nt long) that are encoded by distinct mitochondrial chromosomes, yet the rRNA is continuous. To reconstruct the post-transcriptional maturation pathway of this rRNA, we have catalogued transcript intermediates by deep RNA sequencing and RT-PCR. Gene modules are transcribed separately. Subsequently, transcripts are end-processed, the module-1 transcript is polyuridylated and the module-2 transcript is polyadenylated. The two modules are joined via trans-splicing that retains at the junction similar to 26 uridines, resulting in an extent of insertion RNA editing not observed before in any system. The A-tail of trans-spliced molecules is shorter than that of mono-module 2, and completely absent from mitoribosome-associated mt-LSU rRNA. We also characterize putative antisense transcripts. Antisense-mono-modules corroborate bi-directional transcription of chromosomes...

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...

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...

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...

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...

Identification and characterization of RNA guanine-quadruplex binding proteins
von Hacht, A; Seifert, O; Menger, M; Schutze, T; Arora, A; Konthur, Z; Neubauer, P; Wagner, A; Weise, C; Kurreck, J
Nucleic Acids Res. 2014, 42, 6630-6644
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Guanine quadruplex (G-quadruplex) motifs in the 5' untranslated region (5'-UTR) of mRNAs were recently shown to influence the efficiency of translation. In the present study, we investigate the interaction between cellular proteins and the G-quadruplexes located in two mRNAs (MMP16 and ARPC2). Formation of the G-quadruplexes was confirmed by biophysical characterization and the inhibitory activity on translation was shown by luciferase reporter assays. In experiments with whole cell extracts from different eukaryotic cell lines, G-quadruplex-binding proteins were isolated by pull-down assays and subsequently identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The binding partners of the RNA G-quadruplexes we discovered included several heterogenous nuclear ribonucleoproteins, ribosomal proteins, and splicing factors, as well as other proteins that have previously not been described to interact with nucleic acids. While most of the proteins were specific for either of the investigated G-quadruplexes, some of them bound to both motifs...

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...

Secondary structure and domain architecture of the 23S and 5S rRNAs
Petrov, AS; Bernier, CR; Hershkovits, E; Xue, YZ; Waterbury, CC; Hsiao, CL; Stepanov, VG; Gaucher, EA; Grover, MA; Harvey, SC; Hud, NV; Wartell, RM; Fox, GE; Williams, LD
Nucleic Acids Res. 2013, 41, 7522-7535
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We present a de novo re-determination of the secondary (2 degrees) structure and domain architecture of the 23S and 5S rRNAs, using 3D structures, determined by X-ray diffraction, as input. In the traditional 2 degrees structure, the center of the 23S rRNA is an extended single strand, which in 3D is seen to be compact and double helical. Accurately assigning nucleotides to helices compels a revision of the 23S rRNA 2 degrees structure. Unlike the traditional 2 degrees structure, the revised 2 degrees structure of the 23S rRNA shows architectural similarity with the 16S rRNA. The revised 2 degrees structure also reveals a clear relationship with the 3D structure and is generalizable to rRNAs of other species from all three domains of life. The 2 degrees structure revision required us to reconsider the domain architecture. We partitioned the 23S rRNA into domains through analysis of molecular interactions, calculations of 2D folding propensities and compactness. The best domain model for the 23S rRNA contains seven domains, not six as previously ascribed...

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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