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Ribosome profiling: a Hi‐Def monitor for protein synthesis at the genome‐wide scale

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Ribosome profiling or ribo‐seq is a new technique that provides genome‐wide information on protein synthesis (GWIPS) in vivo. It is based on the deep sequencing of ribosome protected mRNA fragments allowing the measurement of ribosome density along all RNA molecules present in the cell. At the same time, the high resolution of this technique allows detailed analysis of ribosome density on individual RNAs. Since its invention, the ribosome profiling technique has been utilized in a range of studies in both prokaryotic and eukaryotic organisms. Several studies have adapted and refined the original ribosome profiling protocol for studying specific aspects of translation. Ribosome profiling of initiating ribosomes has been used to map sites of translation initiation. These studies revealed the surprisingly complex organization of translation initiation sites in eukaryotes. Multiple initiation sites are responsible for the generation of N‐terminally extended and truncated isoforms of known proteins as well as for the translation of numerous open reading frames (ORFs), upstream of protein coding ORFs. Ribosome profiling of elongating ribosomes has been used for measuring differential gene expression at the level of translation, the identification of novel protein coding genes and ribosome pausing. It has also provided data for developing quantitative models of translation. Although only a dozen or so ribosome profiling datasets have been published so far, they have already dramatically changed our understanding of translational control and have led to new hypotheses regarding the origin of protein coding genes. WIREs RNA 2013, 4:473–490. doi: 10.1002/wrna.1172 This article is categorized under: Translation > Ribosome Structure/Function Translation > Translation Mechanisms Translation > Translation Regulation RNA Methods > RNA Analyses in Cells

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The emplacement of genome‐wide information on protein synthesis (GWIPS) and the role of ribo‐seq in characterizing the molecular status of the cell.
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Detection of protein isoforms with alternative N‐termini. Panel (a) shows an N‐terminally extended isoform of the human RND3 gene which has an in‐frame CUG initiating codon. Panel (b) shows a truncated isoform of the human CLK3 gene which was found to initiate at an AUG codon downstream of the annotated AUG start codon (Reprinted with permission from Ref . Copyright 2012 National Academy of Sciences USA.)
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The ribosome initiating profiles [harringtonine (Harr) and lactimidomycin (LTM)] and elongating profiles [cycloheximide (CHX)] for the HCMV genes UL38 (a) and UL10 (b). The two ribosome profiling approaches aided the identification of internal initiation sites in both genes, with an N‐terminally truncated translation product for UL38 and a previously unknown out‐of‐frame translated ORF contained within the UL10 gene (Reprinted with permission from Ref . Copyright 2012 AAAS.)
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The utilization of triplet periodicity for detecting transitions in translated reading frames. Panel (a) shows the absolute number of RPFs aligning to each subcodon position for the coding region of human antizyme 1 (OAZ1) mRNA. The location of the programmed ribosomal frameshift site is indicated by a broken black line. Panel (b) shows the distribution of the number of RPFs aligning to different sub‐codon positions, upstream of the frameshift site (left) and downstream (right). It can be seen that the sub‐codon position with the lowest RPF count shifts from the second to the third upon ribosomal frameshifting which is consistent with the +1 directionality of the programmed ribosomal frameshift utilized by OAZ1 in its expression (Reprinted with permission from Ref . Copyright 2012 Cold Spring Harbor Laboratory Press)
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Subcodon ribosome profiles for human NPAS2 (left‐hand side) and THAP7 (right‐hand side) mRNAs. The triplet periodicity of ribosome profiles allows the discrimination of the translated reading frame by separating footprints into subcodon positions depending on the phase of their 5′‐ends (a). In both cases, the subcodon profiles exhibit the pattern consistent with translation of alternative ORFs (highlighted in pink in b). The functionality of these two ORFs is supported by deep phylogenetic conservation that is evident from the comparative sequence alignments shown in (c) (Reprinted with permission from Ref . Copyright 2012 Cold Spring Harbor Laboratory Press)
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The increased ribosome density at known sites of ribosome stalling: secM (a) and tnaC (b) in E. coli; mifM (c) in B. subtilis; and Xbp1 in Mus musculus (d). Black arrows indicate the locations of known ribosome pause sites (a–c: Reprinted with permission from Ref . Copyright 2012 Mcmillan Publishers Ltd; d: Reprinted with permission from Ref . Copyright 2011 Elsevier)
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Examples of temporal translational control. Panel (a) shows the expression levels of the adjacent SPS1 and SPS2 genes at different stages of meiosis in S. cerevisiae. The mRNA levels are consistent throughout all stages of meiosis. However, the ribosome profiling data for SPS1 shows strong temporal translational regulation while SPS2 does not (Reprinted with permission from Ref . Copyright 2012 AAAS). Panel (b) provides a heatmap of the ribosome density of viral genes clustered according to expression levels at 5, 24, and 72 h after the infection of human foreskin fibroblasts with cytomegalovirus (Reprinted with permission from Ref . Copyright 2012 AAAS)
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Ribo‐seq (red) and mRNA‐seq (green) coverage plots for the S. cerevisiae genome locus containing ABP140, MET7, SSP2, and PUS7 genes obtained with GWIPS‐viz (http://gwips.ucc.ie/) using data from Ref . Under starvation conditions (right), ABP140, MET7 and PUS7 are transcribed, but not translated.
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Two main ribo‐seq strategies: ribosome profiling of elongating ribosomes (top, blue arrow) and ribosome profiling of initiating ribosomes (bottom, light‐pink arrow). In both cases, the freezing of ribosomes at specific stages of translation is followed by the degradation of mRNA unprotected by ribosomes and subsequent preparation of ribosome footprint cDNA libraries and their sequencing. The right‐hand side of the figure illustrates how the data obtained with these ribo‐seq techniques can be analyzed for the identification of uORFs (shown as pink areas in the left plot), protein isoforms with alternative N‐termini (middle plot), and nORFs embedded within annotated coding regions and recoding events (far‐right plot).
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Outline of the major steps of the ribosome profiling protocol as described in Ingolia et al. The experimental part of the protocol requires 7 days. Modifications of the protocol have been made in several other studies and commercial kits for ribosome profiling are currently available.
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RNA Methods > RNA Analyses in Cells
Translation > Ribosome Structure/Function
Translation > Translation Mechanisms
Translation > Translation Regulation

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