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Boris Zinshteyn, Maria F Rojas Duran, Wendy V Gilbert
Translational control of gene expression plays essential roles in cellular stress responses and organismal development by enabling rapid, selective, and localized control of protein production. Translational regulation depends on context-dependent differences in the protein output of mRNAs, but the key mRNA features that distinguish efficiently translated mRNAs are largely unknown. Here we comprehensively determined the RNA-binding preferences of the initiation factor eIF4G to assess whether this core translation initiation factor has intrinsic sequence preferences that may contribute to preferential translation of specific mRNAs...
May 25, 2017: RNA
Sara Silva, David Homolka, Ramesh S Pillai
Ribonucleases catalyse maturation of functional RNAs or mediate degradation of cellular transcripts, activities that are critical for gene expression control. Here we identify a previously uncharacterized mammalian nuclease family member NEF-sp [RNA exonuclease 5 (REXO5) or LOC81691] as a testis-specific factor. Recombinant human NEF-sp demonstrates a divalent metal ion-dependent 3'→5' exoribonuclease activity. This activity is specific to single-stranded RNA substrates and is independent of their length...
May 24, 2017: RNA
Dustin B Ritchie, Tonia R Cappellano, Collin Tittle, Negar Rezajooei, Logan Rouleau, William Ka Sikkema, Michael T Woodside
Programmed ribosomal frameshifting (PRF) in HIV-1 is thought to be stimulated by a hairpin in the mRNA, although a pseudoknot-like triplex has also been proposed. Because the conformational dynamics of the stimulatory structure under tension applied by the ribosomal helicase during translation may play an important role in PRF, we used optical tweezers to apply tension to the HIV stimulatory structure and monitor its unfolding and refolding dynamics. The folding and unfolding kinetics and energy landscape of the hairpin were measured by ramping the force on the hairpin up and down, providing a detailed biophysical characterisation...
May 18, 2017: RNA
Mauro D Locati, Johanna F B Pagano, Geneviève Girard, Wim A Ensink, Marina van Olst, Selina van Leeuwen, Ulrike Nehrdich, Herman P Spaink, Han Rauwerda, Martijs J Jonker, Rob J Dekker, Timo M Breit
There is mounting evidence that the ribosome is not a static translation machinery, but a cell-specific, adaptive system. Ribosomal variations have mostly been studied at the protein level, even though the essential transcriptional functions are primarily performed by rRNAs. At the RNA level, oocyte-specific 5S rRNAs are long known for Xenopus. Recently, we described for zebrafish a similar system in which the sole maternal-type 5S rRNA present in eggs, is replaced completely during embryonic development by a somatic-type...
May 12, 2017: RNA
Yinzhou Zhu, Stephan P Pirnie, Gordon G Carmichael
Ribose methylation (2'-O methylation, 2'-OMe) occurs at high frequencies in rRNAs and other small RNAs, and is carried out using a shared mechanism across eukaryotes and archaea. As RNA modifications are important for ribosome maturation, and alterations in these modifications are associated with cellular defects and diseases, it is important to characterize the landscape of 2'-O methylation. Here we report the development of a highly sensitive and accurate method for ribose methylation detection using next generation sequencing...
May 11, 2017: RNA
Marco Nousch, Ryuji Minasaki, Christian R Eckmann
In biology, the role of many enzymes extends beyond their dedicated catalytic activity by fulfilling important cellular roles in a catalysis-independent fashion. In this aspect, little is known about 3'-end RNA-modifying enzymes that belong to the class of nucleotidyl transferases. Among these are non-canonical poly(A) polymerases, a group of evolutionary conserved enzymes that are critical for gene expression regulation, by adding adenosines to the 3'end of mRNA targets. In this study, we investigate whether the functions of the cytoplasmic poly(A) polymerase (cytoPAP) GLD-2 in C...
May 10, 2017: RNA
Julia Ye, Hu Jin, Aleksandr Pankov, Jun Song, Robert Blelloch
While years of investigation have elucidated many aspects of embryonic stem cell (ESC) regulation, the contributions of post-transcriptional and translational mechanisms to the pluripotency network remain largely unexplored. In particular, little is known in ESCs about the function of RNA binding proteins (RBPs), the protein agents of post-transcriptional regulation. We performed an unbiased RNAi screen of RBPs in an ESC differentiation assay and identified two related genes, NF45 (Ilf2) and NF90/NF110 (Ilf3), whose knockdown promoted differentiation to an epiblast-like state...
May 9, 2017: RNA
Anne Kruse Hollensen, Rune Thomsen, Rasmus O Bak, Charlotte Christie Petersen, Eva Reumert Ermegaard, Lars Aagaard, Christian Kroun Damgaard, Jacob Giehm Mikkelsen
Our genes are posttranscriptionally regulated by microRNAs (miRNAs) inducing translational suppression and degradation of targeted mRNAs. Strategies to inhibit miRNAs in a spatiotemporal manner in a desired cell type or tissue, or at a desired developmental stage, can be crucial for understanding miRNA function and for pushing forward miRNA suppression as a feasible rationale for genetic treatment of disease. For such purposes, RNA polymerase II (RNApolII)-transcribed Tough Decoy (TuD) miRNA inhibitors are particularly attractive...
May 9, 2017: RNA
Birgit Maria Rogell, Bernd Fischer, Mandy Rettel, Jeroen Krijgsveld, Alfredo Castello, Matthias W Hentze
RNA-binding proteins (RBPs) play essential roles in RNA biology, responding to cellular and environmental stimuli to regulate gene expression. Important advances have helped to determine the (near) complete repertoires of cellular RBPs. However, identification of RBPs associated with specific transcripts remains a challenge. Here, we describe "specific ribonucleoprotein (RNP) capture", a versatile method for the determination of the proteins bound to specific transcripts in vitro and in cellular systems. Specific RNP capture employs UV irradiation to covalently stabilize protein-RNA interactions taking place at "zero distance"...
May 5, 2017: RNA
Michael Hackenberg, David Langenberger, Alexandra Schwarz, Jan Erhart, Michail Kotsyfakis
The hard tick Ixodes ricinus is an important disease vector whose salivary secretions mediate blood feeding success on vertebrate hosts, including humans. Here we describe the expression profiles and downstream analysis of de novo discovered microRNAs (miRNAs) expressed in I. ricinus salivary glands and saliva. Eleven tick-derived libraries were sequenced to produce 67,375,557 Illumina reads. De novo prediction yielded 67 bona-fide miRNAs out of which 35 are currently not present in miRBase. We report for the first time the presence of microRNAs in tick saliva obtaining furthermore molecular indicators that those might be of exosomal origin...
May 4, 2017: RNA
Samuel G Rouleau, Jean-Pierre Sehi Glouzon, Andrea Brumwell, Martin Bisaillon, Jean-Pierre Perreault
MicroRNAs (miRNA) are small non-coding RNAs that repress the translation of their target genes. It has previously been shown that a target's availability to miRNA can be affected by its structure. G-quadruplexes (G4) are non-canonical structures adopted by G-rich nucleic acids which have been shown to have multiple biological functions. In this study, whether or not G4 structures present in the 3' UTRs of mRNAs can hinder miRNA binding was investigated. Putative G4 overlapping with predicted miRNAs binding sites were searched for, and 44,294 hits were found in humans...
May 4, 2017: RNA
Steven Parker, Marcin Fraczek, Jian Wu, Sara Shamsah, Alkisti Manousaki, Kobchai Dungrattanalert, Rogerio Alves de Almeida, Diego Estrada-Rivadeneyra, Walid Omara, Daniela Delneri, Raymond T O'Keefe
Eukaryotic genomes are extensively transcribed generating many different RNAs with no known function. We have constructed 1502 molecular barcoded ncRNA gene deletion strains encompassing 443 ncRNAs in the yeast Saccharomyces cerevisiae as tools for ncRNA functional analysis. This resource includes deletions of small nuclear RNAs (snRNAs), transfer RNAs (tRNAs), small nucleolar RNAs (snoRNAs) and other annotated ncRNAs as well as the more recently identified stable unannotated transcripts (SUTs) and cryptic unstable transcripts (CUTs) whose functions are largely unknown...
May 3, 2017: RNA
Franziska Ottens, Volker Boehm, Christopher R Sibley, Jernej Ule, Niels H Gehring
Nonsense-mediated mRNA decay (NMD) controls gene expression by eliminating mRNAs with premature or aberrant translation termination. Degradation of NMD substrates is initiated by the central NMD factor UPF1, which recruits the endonuclease SMG6 and the deadenylation-promoting SMG5/7 complex. The extent to which SMG5/7 and SMG6 contribute to the degradation of individual substrates and their regulation by UPF1 remain elusive. Here we map transcriptome-wide sites of SMG6-mediated endocleavage via 3' fragment capture and degradome sequencing...
May 1, 2017: RNA
Christopher B Ball, Amanda C Solem, Rita M Meganck, Alain Laederach, Silvia Bv Ramos
ZFP36L2 protein destabilizes AU-rich element-containing transcripts and has been implicated in female fertility. We have shown that only one of three putative AREs within the 3' UTR of murine LHR mRNA, i.e., ARE 2197, is capable of interacting with ZFP36L2. To dissect the structural elements of ARE 2197 that mediate protein-RNA binding, we performed whole-transcript SHAPE-MaP (Selective 2' Hydroxyl Acylation by Primer Extension-Mutational Profiling). The non-functional ARE 2301 contains nucleotides that are strongly base paired, thus do not interact with ZFP36L2...
April 28, 2017: RNA
Lizhen Sun, Jingxiang Zhang, Shi-Jie Chen
Metal ions play critical roles in RNA structure and function. However, web servers and software packages for predicting ion effects in RNA structures is notably scarce. Furthermore, the existing web servers and software packages mainly neglect ion correlation and fluctuation effects, which are potentially important for RNAs. We here report a new web server, MCTBI server (, for the prediction of ion effects for RNA structures. The server is based on the recently developed MCTBI, a model that can account for ion correlation and fluctuation effects for nucleic acids structures and can provide improved predictions for the effects of metal ions, especially for multivalent ions such as Mg2+ effects, as shown by extensive theory-experiment test results...
April 27, 2017: RNA
Shivani Sharma, Neha Nagpal, Prahlad C Ghosh, Ritu Kulshreshtha
miRNAs have emerged as key participants of p53 signaling pathway as they regulate or be regulated by p53. Here, we provide the first study demonstrating direct regulation of an oncogenic miRNA, miR-191, by p53 and existence of a regulatory feedback loop. Using a combination of qRT-PCR, promoter-luciferase and chromatin-immunoprecipitation assays we show that p53 brings about downregulation of miR-191 in breast cancer. miR-191 overexpression brought about inhibition of apoptosis in breast cancer cell lines (MCF7 and ZR-75) as demonstrated by reduction in annexin-V stained cells and caspase 3/7 activity while miR-191 downregulation showed the opposite...
April 27, 2017: RNA
Kelsi A Lindblad, John R Bracht, April E Williams, Laura Landweber
The ciliate Oxytricha trifallax maintains two genomes: a germline genome that is active only during sexual conjugation and a transcriptionally active, somatic genome that derives from the germline via extensive sequence reduction and rearrangement. Previously, we found that long, non-coding (lnc) RNA "templates" - telomere-containing, RNA-cached copies of mature chromosomes - provide the information to program the rearrangement process. Here we used a modified RNA-seq approach to conduct the first genome-wide search for endogenous, telomere-to-telomere RNA transcripts in any eukaryotic genome...
April 27, 2017: RNA
Gary Loughran, Michael T Howard, Andrew E Firth, John F Atkins
Positioning test sequences between fused reporters permits monitoring of both translation levels and framing, before and after the test sequence. Many studies, including those on recoding such as productive ribosomal frameshifting and stop codon readthrough, use distinguishable luciferases or fluorescent proteins, as reporters. Occasional distortions, due to test sequence product interference with the individual reporter activities or stabilities, are here shown to be avoidable by the introduction of tandem StopGo sequences (2A) flanking the test sequence...
April 25, 2017: RNA
Svetlana Deryusheva, Joseph G Gall
The pseudouridine at position 43 in vertebrate U2 snRNA is one of the most conserved posttranscriptional modifications of spliceosomal snRNAs; the equivalent position is pseudouridylated in U2 snRNAs in different phyla including fungi, insects, and worms. Pseudouridine synthase Pus1p acts alone on U2 snRNA to form this pseudouridine in yeast Saccharomyces cerevisiae and mouse. Furthermore, in S. cerevisiae Pus1p is the only pseudouridine synthase for this position. Using an in vivo yeast cell system we tested enzymatic activity of Pus1p from the fission yeast Schizosaccharomyces pombe, the worm Caenorhabditis elegans, the fruit fly Drosophila melanogaster and the frog Xenopus tropicalis...
April 21, 2017: RNA
Irina I Mohorianu, Amanda Bretman, Damian T Smith, Emily Fowler, Tamas Dalmay, Tracey Chapman
Socio-sexual environments have profound effects on fitness. Local sex ratios can alter the threat of sexual competition, to which males respond via plasticity in reproductive behaviours and ejaculate composition. In Drosophila melanogaster, males detect the presence of conspecific mating rivals prior to mating using multiple, redundant sensory cues. Males that respond to rivals gain significant fitness benefits by altering mating duration and ejaculate composition. Here we investigated the underlying genome-wide changes involved...
April 20, 2017: RNA
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