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      Genome-wide assessment of differential translations with ribosome profiling data

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          Abstract

          The closely regulated process of mRNA translation is crucial for precise control of protein abundance and quality. Ribosome profiling, a combination of ribosome foot-printing and RNA deep sequencing, has been used in a large variety of studies to quantify genome-wide mRNA translation. Here, we developed Xtail, an analysis pipeline tailored for ribosome profiling data that comprehensively and accurately identifies differentially translated genes in pairwise comparisons. Applied on simulated and real datasets, Xtail exhibits high sensitivity with minimal false-positive rates, outperforming existing methods in the accuracy of quantifying differential translations. With published ribosome profiling datasets, Xtail does not only reveal differentially translated genes that make biological sense, but also uncovers new events of differential translation in human cancer cells on mTOR signalling perturbation and in human primary macrophages on interferon gamma (IFN-γ) treatment. This demonstrates the value of Xtail in providing novel insights into the molecular mechanisms that involve translational dysregulations.

          Abstract

          The global measurement of ribosome occupancy on mRNAs is commonly used as a proxy in estimating rates of protein synthesis. Here the authors describe Xtail, a computational approach that facilitates the extraction of accurate quantitative insight from ribosome profiling data (Ribo-Seq).

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          Most cited references36

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          Ribosome profiling shows that miR-430 reduces translation before causing mRNA decay in zebrafish.

          MicroRNAs regulate gene expression through deadenylation, repression, and messenger RNA (mRNA) decay. However, the contribution of each mechanism in non-steady-state situations remains unclear. We monitored the impact of miR-430 on ribosome occupancy of endogenous mRNAs in wild-type and dicer mutant zebrafish embryos and found that miR-430 reduces the number of ribosomes on target mRNAs before causing mRNA decay. Translational repression occurs before complete deadenylation, and disrupting deadenylation with use of an internal polyadenylate tail did not block target repression. Lastly, we observed that ribosome density along the length of the message remains constant, suggesting that translational repression occurs by reducing the rate of initiation rather than affecting elongation or causing ribosomal drop-off. These results show that miR-430 regulates translation initiation before inducing mRNA decay during zebrafish development.
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            Ribosome profiling reveals pervasive translation outside of annotated protein-coding genes.

            Ribosome profiling suggests that ribosomes occupy many regions of the transcriptome thought to be noncoding, including 5' UTRs and long noncoding RNAs (lncRNAs). Apparent ribosome footprints outside of protein-coding regions raise the possibility of artifacts unrelated to translation, particularly when they occupy multiple, overlapping open reading frames (ORFs). Here, we show hallmarks of translation in these footprints: copurification with the large ribosomal subunit, response to drugs targeting elongation, trinucleotide periodicity, and initiation at early AUGs. We develop a metric for distinguishing between 80S footprints and nonribosomal sources using footprint size distributions, which validates the vast majority of footprints outside of coding regions. We present evidence for polypeptide production beyond annotated genes, including the induction of immune responses following human cytomegalovirus (HCMV) infection. Translation is pervasive on cytosolic transcripts outside of conserved reading frames, and direct detection of this expanded universe of translated products enables efforts at understanding how cells manage and exploit its consequences. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.
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              Widespread regulation of translation by elongation pausing in heat shock.

              Global repression of protein synthesis is a hallmark of the cellular stress response and has been attributed primarily to inhibition of translation initiation, although this mechanism may not always explain the full extent of repression. Here, using ribosome footprinting, we show that 2 hr of severe heat stress triggers global pausing of translation elongation at around codon 65 on most mRNAs in both mouse and human cells. The genome-wide nature of the phenomenon, its location, and features of protein N termini suggested the involvement of ribosome-associated chaperones. After severe heat shock, Hsp70's interactions with the translational machinery were markedly altered and its association with ribosomes was reduced. Pretreatment with mild heat stress or overexpression of Hsp70 protected cells from heat shock-induced elongation pausing, while inhibition of Hsp70 activity triggered elongation pausing without heat stress. Our findings suggest that regulation of translation elongation in general, and by chaperones in particular, represents a major component of cellular stress responses. Copyright © 2013 Elsevier Inc. All rights reserved.
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                Author and article information

                Journal
                Nat Commun
                Nat Commun
                Nature Communications
                Nature Publishing Group
                2041-1723
                04 April 2016
                2016
                : 7
                : 11194
                Affiliations
                [1 ]MOE Key Laboratory of Bioinformatics, Tsinghua University , Beijing 100084, China
                [2 ]Tsinghua-Peking Joint Center for Life Sciences , Beijing 100084, China
                [3 ]Center for Synthetic and Systems Biology, Tsinghua University , Beijing 100084, China
                [4 ]School of Life Sciences, Tsinghua University , Beijing 100084, China
                [5 ]Joint Graduate Program of Peking–Tsinghua–National Institute of Biological Science, Tsinghua University , Beijing 100084, China
                Author notes
                Article
                ncomms11194
                10.1038/ncomms11194
                4822032
                27041671
                3625eb72-f79b-4f26-8f85-47d3feb45d8d
                Copyright © 2016, Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.

                This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

                History
                : 06 July 2015
                : 29 February 2016
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