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Rei Yoshimoto, Daisuke Kaida, Masaaki Furuno, A Maxwell Burroughs, Shohei Noma, Harukazu Suzuki, Yumi Kawamura, Yoshihide Hayashizaki, Akila Mayeda, Minoru Yoshida
Spliceostatin A (SSA) is a methyl ketal derivative of FR901464, a potent antitumor compound isolated from a culture broth of Pseudomonas sp. no. 2663. These compounds selectively bind to the essential spliceosome component SF3b, a subcomplex of the U2 snRNP, to inhibit pre-mRNA splicing. However, the mechanism of SSA's antitumor activity is unknown. It is noteworthy that SSA causes accumulation of a truncated form of the CDK inhibitor protein p27 translated from CDKN1B pre-mRNA, which is involved in SSA-induced cell-cycle arrest...
October 17, 2016: RNA
Cindo O Nicholson, Matthew B Friedersdorf, Jack D Keene
RNA-binding proteins (RBPs) and non-coding RNAs orchestrate post-transcriptional processes through the recognition of specific sites on targeted transcripts. Thus, understanding the connection between binding to specific sites and active regulation of the whole transcript is essential. Many immunoprecipitation techniques have been developed that identify either whole transcripts or binding sites of RBPs on each transcript using cell lysates. However, none of these methods simultaneously measures the strength of each binding site, and quantifies binding to whole transcripts...
October 14, 2016: RNA
Yuda Wei, Yan Qiu, Yanhao Chen, Gaigai Liu, Yongxian Zhang, Luwei Xu, Qiurong Ding
Multiplex genome engineering in vivo with CRISPR/Cas9 shows great promise as a potential therapeutic approach. The ability to incorporate multiple single guide RNA (sgRNA) cassettes together with Cas9 gene expression in one AAV vector could greatly enhance the efficiency. In a recent Method article, Mefferd et al indicated that small tRNA promoters could be used to drive sgRNA expression to facilitate the construction of a more effective AAV vector. In contrast, we found that when targeting endogenous genomic loci, CRISPR/Cas9 with tRNA promoter-driven sgRNA expression showed much reduced genome editing activity, compared with significant cleavage with U6 promoter-driven sgRNA expression...
October 14, 2016: RNA
Dziyana Hnedzko, Dennis W McGee, Yannis A Karamitas, Eriks Rozners
Sequence-selective recognition of complex RNAs in live cells could find broad applications in biology, biomedical research and biotechnology. However, specific recognition of structured RNA is challenging and generally applicable and effective methods are lacking. Recently, we found that peptide nucleic acids (PNAs) were unusually well suited ligands for recognition of double-stranded RNAs. Herein, we report that 2-aminopyridine (M) modified PNAs and their conjugates with lysine and arginine tripeptides form strong (Ka = 9...
October 14, 2016: RNA
Vera P Pisareva, Andrey V Pisarev
Eukaryotic translation initiation is a complex process involving many components. eIF3 is a scaffold for multiple initiation factors and plays multiple roles in initiation, and DHX29 helicase enhances the formation of the 48S initiation complex on structured mRNAs. Because DHX29 is not a processive helicase, the mechanism underlying its activity is unclear. Here, we show that DHX29 establishes many points of contact with eIF3. In particular, the unique N terminus of DHX29 associates with the RNA recognition motif of eIF3b and the C terminus of the eIF3a subunits of eIF3, and the disruption of either contact impairs DHX29 activity...
October 12, 2016: RNA
Beate Schwer, Fahad Khalid, Stewart Shuman
Saccharomyces cerevisiae Dbr1 is a manganese-dependent RNA debranching enzyme that cleaves the 2'-5' phosphodiester bond of the lariat introns formed during pre-mRNA splicing. Dbr1 is a member of the binuclear metallophosphoesterase enzyme superfamily. We showed previously via alanine scanning that RNA debranching in vivo and in vitro depends on conserved active site residues His13, Asp40, Asn85, His86, His179, His231, and His233. Here, by extending the alanine scan, we added Cys11 to the ensemble of essential active site components...
October 7, 2016: RNA
Pietro Freihofer, Rashid Akbergenov, Youjin Teo, Reda Juskeviciene, Dan I Andersson, Erik C Böttger
Several studies over the last few decades have shown that antibiotic resistance mechanisms frequently confer a fitness cost and that these costs can be genetically ameliorated by intra- or extragenic second-site mutations, often without loss of resistance. Another, much less studied potential mechanism by which the fitness cost of antibiotic resistance could be reduced is via a regulatory response where the deleterious effect of the resistance mechanism is lowered by a physiological alteration that buffers the mutational effect...
October 3, 2016: RNA
Daniel W Binzel, Emil Khisamutdinov, Mario Vieweger, Janice Ortega, Jingyuan Li, Peixuan Guo
RNA nanotechnology is rapidly emerging. Due to advantageous pharmacokinetics and favorable in vivo biodistribution, RNA nanoparticles have shown promise in targeted delivery of therapeutics. RNA nanotechnology applies bottom-up assembly, thus elucidation of the mechanism of interaction between multiple components is of fundamental importance. The tendency of diminishing concern about RNA instability has accelerated by the finding of the novel thermostable three-way junction (3WJ) motif of the phi29 DNA-packaging motor...
September 26, 2016: RNA
Karen M Ruff, Ayesha Muhammad, Phillip J McCown, Ronald R Breaker, Scott A Strobel
The glycine riboswitch often occurs in a tandem architecture, with two ligand-binding domains (aptamers) followed by a single expression platform. Based on previous observations, we hypothesized that "singlet" versions of the glycine riboswitch, which contain only one aptamer domain, are able to bind glycine if appropriate structural contacts are maintained. An initial alignment of 17 putative singlet riboswitches indicated that the single consensus aptamer domain is flanked by a conserved peripheral stem-loop structure...
September 22, 2016: RNA
Amir H Nasiri, Jan Philip Wurm, Carina Immer, Anna Katharina Weickhmann, Jens Wöhnert
Many naturally occurring or artificially created RNAs are capable of binding to guanine or guanine derivatives with high affinity and selectivity. They bind their ligands using very different recognition modes involving a diverse set of hydrogen bonding and stacking interactions. Apparently, the potential structural diversity for guanine, guanosine, and guanine nucleotide binding motifs is far from being fully explored. Szostak and coworkers have derived a large set of different GTP-binding aptamer families differing widely in sequence, secondary structure, and ligand specificity...
September 22, 2016: RNA
Geneviève Di Tomasso, Lisa M Miller Jenkins, Pascale Legault
As part of their normal life cycle, most RNA molecules associate with several proteins that direct their fate and regulate their function. Here, we describe a novel method for identifying proteins that associate with a target RNA. The procedure is based on the ARiBo method for affinity purification of RNA, which was originally developed to quickly purify RNA with high yields and purity under native conditions. The ARiBo method was further optimized using in vitro transcribed RNA to capture RNA-associating proteins from cellular extracts with high yields and low background protein contamination...
September 22, 2016: RNA
Wei Zhang, Mingyi Xie, Mei-Di Shu, Joan A Steitz, Daniel DiMaio
The proximity ligation assay (PLA) is an immune staining method that detects protein-protein interactions in fixed cells. We describe here RNA-PLA, a simple adaptation of this technology that allows the detection of specific RNA-protein interactions in fixed cells by using a DNA oligonucleotide that hybridizes to a target RNA in combination with an antibody that recognizes the protein bound to the target RNA. Stable and transient RNA-protein interactions can be readily detected by generation of a fluorescent signal in discrete compartments in intact fixed cells with high specificity...
September 22, 2016: RNA
Knut I Kristiansen, Ragnhild Weel-Sneve, James A Booth, Magnar Bjørås
Protein translation can be affected by changes in the secondary structure of mRNA. The dinQ gene in Escherichia coli encodes a primary transcript (+1) that is inert to translation. Ribonucleolytic removal of the 44 first nucleotides converts the +1 transcript into a translationally active form, but the mechanism behind this structural change is unknown. Here we present experimental evidence for a mechanism where alternative RNA secondary structures in the two dinQ mRNA variants affect translation initiation by mediating opening or closing of the ribosome binding sequence...
September 20, 2016: RNA
Aaron Coey, Kevin Larsen, Joseph D Puglisi, Elisabetta Viani Puglisi
Reverse transcription is a key process in the early steps of HIV infection. This process initiates within a specific complex formed by the 5' UTR of the HIV genomic RNA (vRNA) and a host primer tRNA(Lys) 3 Using nuclear magnetic resonance (NMR) spectroscopy and single-molecule fluorescence spectroscopy, we detect two distinct conformers adopted by the tRNA/vRNA initiation complex. We directly show that an interaction between the conserved 8-nucleotide viral RNA primer activation signal (PAS) and the primer tRNA occurs in one of these conformers...
September 9, 2016: RNA
Wesley C Clark, Molly E Evans, Dan Dominissini, Guanqun Zheng, Tao Pan
Eukaryotic transfer RNAs contain on average 14 modifications. Investigations of their biological functions require the determination of the modification sites and the dynamic variations of the modification fraction. Base methylation represents a major class of tRNA modification. Although many approaches have been used to identify tRNA base methylations, including sequencing, they are generally qualitative and do not report the information on the modification fraction. Dynamic mRNA modifications have been shown to play important biological roles; yet, the extent of tRNA modification fractions has not been reported systemically...
September 9, 2016: RNA
Mridusmita Saikia, Xiaoyun Wang, Yuanhui Mao, Ji Wan, Tao Pan, Shu-Bing Qian
It is common wisdom that codon usage bias has evolved in the selection for efficient translation, in which highly expressed genes are encoded predominantly by optimal codons. However, a growing body of evidence suggests regulatory roles for non-optimal codons in translation dynamics. Here we report that in mammalian cells, non-optimal codons play a critical role in promoting selective mRNA translation during amino acid starvation. During starvation, in contrast to genes encoding ribosomal proteins whose translation is highly sensitive to amino acid deprivation, translation of genes involved in the cellular protein degradation pathways remains unaffected...
September 9, 2016: RNA
Patrick E Lackey, Joshua D Welch, William F Marzluff
The replication-dependent histone mRNAs end in a stem-loop instead of the poly(A) tail present at the 3' end of all other cellular mRNAs. Following processing, the 3' end of histone mRNAs is trimmed to 3 nucleotides (nt) after the stem-loop, and this length is maintained by addition of nontemplated uridines if the mRNA is further trimmed by 3'hExo. These mRNAs are tightly cell-cycle regulated, and a critical regulatory step is rapid degradation of the histone mRNAs when DNA replication is inhibited. An initial step in histone mRNA degradation is digestion 2-4 nt into the stem by 3'hExo and uridylation of this intermediate...
September 8, 2016: RNA
Yu Guo, Katherine Alexander, Andrew G Clark, Andrew Grimson, Haiyuan Yu
Analysis of transcription regulatory networks has revealed many principal features that govern gene expression regulation. MicroRNAs (miRNAs) have emerged as another major class of gene regulators that influence gene expression post-transcriptionally, but there remains a need to assess quantitatively their global roles in gene regulation. Here, we have constructed an integrated gene regulatory network comprised of transcription factors (TFs), miRNAs, and their target genes and analyzed the effect of regulation on target mRNA expression, target protein expression, protein-protein interaction, and disease association...
September 7, 2016: RNA
Dennis Walczyk, Dagmar K Willkomm, Roland K Hartmann
Ribonuclease P is the ubiquitous endonuclease that generates the mature 5'-ends of precursor tRNAs. In bacteria, the enzyme is composed of a catalytic RNA (∼400 nucleotides) and a small essential protein subunit (∼13 kDa). Most bacterial RNase P RNAs (P RNAs) belong to the architectural type A; type B RNase P RNA is confined to the low-G+C Gram-positive bacteria. Here we demonstrate that the L5.1-L15.1 intradomain contact in the catalytic domain of the prototypic type B RNase P RNA of Bacillus subtilis is crucial for adopting a compact functional conformation: Disruption of the L5...
September 7, 2016: RNA
Yuan Yue, Yun Yang, Lanzhi Dai, Guozheng Cao, Ran Chen, Weiling Hong, Baoping Liu, Yang Shi, Yijun Meng, Feng Shi, Mu Xiao, Yongfeng Jin
No abstract text is available yet for this article.
October 2016: RNA
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