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March 2015


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...

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...

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...

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...

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...

Nova1 is a master regulator of alternative splicing in pancreatic beta cells
Villate, O; Turatsinze, JV; Mascali, LG; Grieco, FA; Nogueira, TC; Cunha, DA; Nardelli, TR; Sammeth, M; Salunkhe, VA; Esguerra, JLS; Eliasson, L; Marselli, L; Marchetti, P; Eizirik, DL
Nucleic Acids Res. 2014, 42, 11818-11830
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Alternative splicing (AS) is a fundamental mechanism for the regulation of gene expression. It affects more than 90% of human genes but its role in the regulation of pancreatic beta cells, the producers of insulin, remains unknown. Our recently published data indicated that the 'neuron-specific' Nova1 splicing factor is expressed in pancreatic beta cells. We have presently coupled specific knockdown (KD) of Nova1 with RNA-sequencing to determine all splice variants and downstream pathways regulated by this protein in beta cells. Nova1 KD altered the splicing of nearly 5000 transcripts. Pathway analysis indicated that these genes are involved in exocytosis, apoptosis, insulin receptor signaling, splicing and transcription. In line with these findings, Nova1 silencing inhibited insulin secretion and induced apoptosis basally and after cytokine treatment in rodent and human beta cells. These observations identify a novel layer of regulation of beta cell function, namely AS controlled by key splicing regulators such as Nova1.

Tumor-targeted in vivo gene silencing via systemic delivery of cRGD-conjugated siRNA
Liu, XX; Wang, W; Samarsky, D; Liu, L; Xu, Q; Zhang, WQ; Zhu, GZ; Wu, P; Zuo, XL; Deng, HL; Zhang, JJ; Wu, ZM; Chen, XH; Zhao, LF; Qiu, ZY; Zhang, ZY; Zeng, QY; Yang, W; Zhang, BL; Ji, AM
Nucleic Acids Res. 2014, 42, 11805-11817
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RNAi technology is taking strong position among the key therapeutic modalities, with dozens of siRNA-based programs entering and successfully progressing through clinical stages of drug development. To further explore potentials of RNAi technology as therapeutics, we engineered and tested VEGFR2 siRNA molecules specifically targeted to tumors through covalently conjugated cyclo(Arg-Gly-Asp-d-Phe-Lys[PEG-MAL]) (cRGD) peptide, known to bind alpha v beta 3 integrin receptors. cRGD-siRNAs were demonstrated to specifically enter and silence targeted genes in cultured alpha v beta 3 positive human cells (HUVEC). Microinjection of zebrafish blastocysts with VEGFR2 cRGD-siRNA resulted in specific inhibition of blood vessel growth. In tumor-bearing mice, intravenously injected cRGD-siRNA molecules generated no innate immune response and bio-distributed to tumor tissues. Continuous systemic delivery of two different VEGFR2 cRGD-siRNAs resulted in downregulation of corresponding mRNA (55 and 45%) and protein (65 and 45%) in tumors...

Solution structure of the YTH domain in complex with N6-methyladenosine RNA: a reader of methylated RNA
Theler, D; Dominguez, C; Blatter, M; Boudet, J; Allain, FHT
Nucleic Acids Res. 2014, 42, 13911-13919
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N(6)A methylation is the most abundant RNA modification occurring within messenger RNA. Impairment of methylase or demethylase functions are associated with severe phenotypes and diseases in several organisms. Beside writer and eraser enzymes of this dynamic RNA epigenetic modification, reader proteins that recognize this modification are involved in numerous cellular processes. Although the precise characterization of these reader proteins remains unknown, preliminary data showed that most potential reader proteins contained a conserved YT521-B homology (YTH) domain. Here we define the YTH domain of rat YT521-B as a N-6-methylated adenosine reader domain and report its solution structure in complex with a N-6-methylated RNA. The structure reveals a binding preference for NGANNN RNA hexamer and a deep hydrophobic cleft for m(6)A recognition. These findings establish a molecular function for YTH domains as m(6)A reader domains and should guide further studies into the biological functions of YTH-containing proteins in m(6)A recognition.

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.

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