Ribosome profiling with translation inhibitors reveals pervasive translation in murine ES cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE67741 | GEO | 2016/01/12
SECONDARY ACCESSION(S): PRJNA280809
REPOSITORIES: GEO
Similar Datasets
Project description:Ribosome profiling with translation inhibitors reveals pervasive translation in murine ES cells. Ribosome profiling or stranded RNAseq (ribominus) with murine embryonic stem cells treated with either DMD-pateamineA, Puromycin, Harringtonine or vehicle (no drug control). Two replicates per condition.
2016-01-12 | E-GEOD-67741 | biostudies-arrayexpress
Project description:Ribosome profiling suggests that ribosomes occupy many regions of the transcriptome thought to be non-coding, including 5' UTRs and 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: co-purification 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 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 to understand how cells manage and exploit its consequences. Ribosome profiling to verify that true ribosome footprints shift in response to different elongation inhibitors (CHX vs Emetine) and co-purify with an affinity-tagged large ribosomal subunit (bound vs input)
2014-08-21 | E-GEOD-60095 | biostudies-arrayexpress
Project description:Ribosome profiling suggests that ribosomes occupy many regions of the transcriptome thought to be non-coding, including 5' UTRs and 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: co-purification 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 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 to understand how cells manage and exploit its consequences.
2014-08-21 | GSE60095 | GEO
Project description:Ribosome Profiling Reveals Pervasive Translation Outside of Annotated Protein-Coding Genes
| PRJNA257463 | ENA
Project description:Ribosome profiling and high-throughput sequencing provide unprecedented opportunities for the analysis of mRNA translation. Using this novel method, several studies have demonstrated the widespread role of short upstream reading frames in translational control as well as slower elongation at the beginning of open reading frames in response to stress. Based on the initial studies, the importance of adding or omitting translation inhibitors, such as cycloheximide, was noted as it markedly affected ribosome coverage profiles. For that reason, many recent studies omitted translation inhibitors in the culture medium. Here, we investigate the influence of ranging cycloheximide concentrations on ribosome profiles in Saccharomyces cerevisiae and demonstrate that increasing the drug concentration can overcome some of the artifacts. We subjected cells to various manipulations and show that neither oxidative stress nor heat shock nor amino acid starvation affect translation elongation. Instead, the observations in the initial studies are the result of cycloheximide-inflicted artifacts. Likewise, we find little support for short upstream reading frames to be involved in wide-spread protein synthesis regulation under stress conditions. Our study highlights the need for better standardization of ribosome profiling methods. Ranging concentrations of cycloheximide and various stress contitions were tested with Ribo-seq
2014-08-01 | E-GEOD-59573 | biostudies-arrayexpress
Project description:Pervasive Translation in Mycobacterium tuberculosis
| PRJEB46152 | ENA
Project description:The vast majority of bacterial open reading frames (ORFs) are identified by automated prediction algorithms. However, these algorithms fail to identify ORFs that deviate from the canonical features of ORFs such as a length of >50 codons, and the presence of an upstream Shine-Dalgarno sequence. Here, we use ribosome profiling approaches to experimentally identify actively translated ORFs in Mycobacterium tuberculosis. Most of the ORFs we identify have not been previously described, indicating that the M. tuberculosis transcriptome is pervasively translated. Moreover, the newly described ORFs are predominantly short, with many encoding proteins of ≤50 amino acids. The codon usage of the newly discovered ORFs suggests that most are not undergoing purifying selection, and hence are unlikely to contribute to cell fitness. Nevertheless, we identify ~90 new ORFs, with a median length of 52 codons, that bear the hallmarks of purifying selection. Thus, our data suggest that pervasive translation of short ORFs serves as a rich source for the evolution of new functional proteins
2021-06-30 | E-MTAB-10695 | biostudies-arrayexpress
Project description:Ribosome profiling and high-throughput sequencing provide unprecedented opportunities for the analysis of mRNA translation. Using this novel method, several studies have demonstrated the widespread role of short upstream reading frames in translational control as well as slower elongation at the beginning of open reading frames in response to stress. Based on the initial studies, the importance of adding or omitting translation inhibitors, such as cycloheximide, was noted as it markedly affected ribosome coverage profiles. For that reason, many recent studies omitted translation inhibitors in the culture medium. Here, we investigate the influence of ranging cycloheximide concentrations on ribosome profiles in Saccharomyces cerevisiae and demonstrate that increasing the drug concentration can overcome some of the artifacts. We subjected cells to various manipulations and show that neither oxidative stress nor heat shock nor amino acid starvation affect translation elongation. Instead, the observations in the initial studies are the result of cycloheximide-inflicted artifacts. Likewise, we find little support for short upstream reading frames to be involved in wide-spread protein synthesis regulation under stress conditions. Our study highlights the need for better standardization of ribosome profiling methods.
2014-08-01 | GSE59573 | GEO
Project description:Open reading frame (ORF) boundaries in bacterial genomes have largely been drawn by gene prediction algorithms. However, these algorithms often fail to predict ORFs with non-canonical features, including those that are short, overlapping, or lack 5’ UTRs. Recent developments in genome-scale mapping of translation have facilitated the empirical identification of open reading frames (ORFs). Here, we use ribosome profiling approaches to map initiating and elongating ribosomes in Mycobacterium tuberculosis. Thus, we identify over 1,000 novel ORFs, revealing that much of the M. tuberculosis genome encodes proteins in overlapping reading frames, and/or on both strands. Most of the novel ORFs are short (sORFs), impeding their identification by traditional methods. The strong codon bias that characterizes annotated mycobacterial ORFs is not evident in the aggregate novel sORFs, and hence most are unlikely to encode functional proteins. Thus, our data suggest that bacterial transcriptomes are subject to pervasive translation that occurs as a result of the relatively low specificity requirements of initiating ribosomes. We speculate that the inefficiency of expressing spurious sORFs may be offset by positive contributions to M. tuberculosis biology through cis and trans regulatory activities of a small subset.
2019-06-20 | E-MTAB-8039 | biostudies-arrayexpress
Project description:Pervasive translation is a widespread phenomenon that plays an important role in de novo gene birth; however, its underlying mechanisms remain unclear. Based on multiple Ribosome Profiling datasets, we investigated the translational landscape of coding and noncoding regions of yeast. Therefore, we developed a new representation framework which allows the visual and comprehensive representation of the diversity of translation behaviors in yeast coding and noncoding regions. We show that if coding regions are restricted to specific regions of the translation landscape, noncoding regions are associated with a wide diversity of translation behaviors and, in contrast, populate the entire yeast translational landscape. In particular, we reveal that noncoding regions are associated with canonical translation signals but also with novel categories of translation events absent from coding regions, and which seem to be a hallmark of pervasive translation. Notably, we report thousands of translated noncoding ORFs among which, 256 led to detectable products with Mass Spectrometry while being characterized by canonical but also non-canonical translation signals. Finally, we show that the translation behavior of noncoding ORFs is not explained by features related to the emergence of function, but is rather determined by the translation start codon and the codon distribution in the three competing RNA frames. Overall, our results enable us to propose a topology of the pervasive translation landscape of a species, and open the way to future comparative analyses of this translation landscape under different conditions.
2024-09-04 | PXD040766 | Pride