Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Ribosome profiling data obtained from HEK293T cells 30 minutes after treatment with arsenite to a final concentration of 40 M-BM-5M

ABSTRACT: Eukaryotic cells rapidly reduce protein synthesis in response to various stress conditions. This can be achieved by the phosphorylation-mediated inactivation of a key translation initiation factor, eIF2. However, the persistent translation of certain mRNAs is required for deployment of an adequate stress response. We carried out ribosome profiling of cultured human cells under conditions of severe stress induced with sodium arsenite. Although this led to a ~4.5-fold general translational repression, the protein coding ORFs of certain individual mRNAs exhibited resistance to the inhibition. Nearly all resistant transcripts possess at least one efficiently translated uORF that repress translation of the main coding ORF under normal conditions. Site specific mutagenesis of two identified stress resistant mRNAs (PPP1R15B and IFRD1) demonstrated that a single uORF is sufficient for eIF2-mediated translation control in both cases. Phylogenetic analysis suggests that at least two regulatory uORFs (namely in SLC35A4 and MIEF1) encode functional protein products. Ribosome profiling of sodium arsenite treated cells for examination of translational response to induction of eIF2 phosphoylation

ORGANISM(S): Homo sapiens

SUBMITTER: Pavel Baranov

PROVIDER: E-GEOD-55195 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Translation of 5' leaders is pervasive in genes resistant to eIF2 repression.

Andreev Dmitry E DE O'Connor Patrick B F PB Fahey Ciara C Kenny Elaine M EM Terenin Ilya M IM Dmitriev Sergey E SE Cormican Paul P Morris Derek W DW Shatsky Ivan N IN Baranov Pavel V PV

eLife 20150126

Eukaryotic cells rapidly reduce protein synthesis in response to various stress conditions. This can be achieved by the phosphorylation-mediated inactivation of a key translation initiation factor, eukaryotic initiation factor 2 (eIF2). However, the persistent translation of certain mRNAs is required for deployment of an adequate stress response. We carried out ribosome profiling of cultured human cells under conditions of severe stress induced with sodium arsenite. Although this led to a 5.4-fo ...[more]

PMID: 25621764

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Project description:Long noncoding RNA (lncRNA) refers to the family of RNA transcripts with more than 200 nucleotides in length, but cannot encode proteins. lincRNA (long intergenic noncoding RNA) is a subset of lncRNA that do not overlap with known genes. Increasing evidences have shown that some of these transcripts do in fact contain open reading frames (ORFs) to code short peptides, and do have significant functional roles within the cells. However, many of these peptides remain unannotated and uncharacterized. This study proposes a workflow integrating proteomics, transcriptomics and bioinformatics specifically for lincRNA-encoded peptide discovery. The workflow was tested on the mouse kidney inner medulla (IM), a region that contains the collecting duct system responsible for regulated water transport. In brief, short peptides (from 2 to 20 kDa) were enriched by tricine protein gel and in-gel trypsinized into peptides, then analyzed using high resolution mass spectrometry. However, to match mass fragment ion spectra to peptide sequences requires a reference peptide sequence database which are not available for the noncoding transcripts, and must be generated de novo in the sample of interest. We modified the RNA-Seq mapping workflow by filtering out coding reads first to better quantitate the noncoding transcript expressions. Also, a rule-based ORF prediction was implemented to select one best predicted ORF per noncoding transcript to construct the peptide library. Candidates were further evaluated using several quality control criteria and bioinformatics tools. Three candidates, conserved in rat and human, passed all criteria, maybe truly novel coding genes. In summary, we present a workflow based on the modern transcriptomics and proteomics technologies for lincRNA-encoded peptide discovery. A computational challenge is to generate a hypothetical lincRNA-encoded peptide database for peptide-mass spectra matching. With this workflow, we discovered three previously unannotated peptides in the mouse kidney inner medulla. The same workflow can be applied in any cell or tissue type of interest to quickly advance this research field.

Dataset Information

Ribosome profiling data obtained from HEK293T cells 30 minutes after treatment with arsenite to a final concentration of 40 M-BM-5M

Publications

Translation of 5' leaders is pervasive in genes resistant to eIF2 repression.

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