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Genome-wide identification of the translation ability of the RNC-SRP complex [RIBO-seq]

ABSTRACT: Ribosome profiling (Ribo-seq) analysis to identify number of ribosome-protected reads from CBC-associated mRNAs or eIF4E-associated mRNAs encoding either a signal sequence or transmembrane domain (TMD)

ORGANISM(S): Homo sapiens

PROVIDER: GSE245550 | GEO | 2024/04/12

REPOSITORIES: GEO

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Genome-wide identification of the translation ability of the RNC-SRP complex [RNA-seq]

Project description:Ribosome profiling (Ribo-seq) analysis to identify number of ribosome-protected reads from CBC-associated mRNAs or eIF4E-associated mRNAs encoding either a signal sequence or transmembrane domain (TMD)

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Genome-wide identification of the translation ability of the RNC-SRP complex [RIBO-seq]

Project description:Genome-wide identification of the translation ability of the RNC-SRP complex [RIBO-seq]

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Nascent Ribo-Seq measures ribosomal loading time and reveals kinetic impact on ribosome density

Project description:mRNAs are generally assumed to be loaded instantly with ribosomes upon entry into the cytoplasm. To measure ribosome density on nascent mRNA, we developed nascent Ribo-Seq (nRibo-Seq) by combining Ribo-Seq with progressive 4-thiouridine labelling. In mouse macrophages, we experimentally determined, for the first time, the lag between the appearance of nascent RNA and its association with ribosomes, which was calculated to be 20 - 22 min for bulk mRNA, and approximated the time it takes for mRNAs to be fully loaded with ribosomes to be 41 - 44 min. Notably, ribosomal loading time is adapted to gene function as rapid loading was observed with highly regulated genes. The lag and ribosomal loading time correlate positively with ORF size and mRNA half-life, and negatively with tRNA adaptation index. Similar results were obtained in mouse embryonic stem cells, where the lag in ribosome loading was even more pronounced with 35 - 38 min. We validated our measurements after stimulation of macrophages with lipopolysaccharide, where the lag between cytoplasmic and translated mRNA leads to uncoupling between input and ribosome-protected fragments. Uncoupling is stronger for mRNAs with long ORFs or half-lives, a finding we also confirmed at the level of protein production by nascent chain proteomics. As a consequence of the lag in ribosome loading, ribosome density measurements are distorted when performed under conditions where mRNA levels are far from steady state expression, and transcriptional changes affect ribosome density in a passive way. This study uncovers an unexpected and considerable lag in ribosome loading, and provides guidelines for the interpretation of Ribo-Seq data taking passive effects on ribosome density into account.

2021-06-22 | PXD026828 | Pride

Genome-wide identification of the translation ability of the RNC-SRP complex

Project description:This SuperSeries is composed of the SubSeries listed below.

2024-04-12 | GSE245551 | GEO

Ribo-Seq timecourse in RAW264.7 cells upon LPS treatment

Project description:mRNAs are generally assumed to be loaded instantly with ribosomes upon entry into the cytoplasm. To measure ribosome density on nascent mRNA, we developed nascent Ribo-Seq (nRibo-Seq) by combining Ribo-Seq with progressive 4-thiouridine labelling. In mouse macrophages, we experimentally determined, for the first time, the lag between the appearance of nascent RNA and its association with ribosomes, which was calculated to be 20 - 22 min for bulk mRNA, and approximated the time it takes for mRNAs to be fully loaded with ribosomes to be 41 - 44 min. Notably, ribosomal loading time is adapted to gene function as rapid loading was observed with highly regulated genes. The lag and ribosomal loading time correlate positively with ORF size and half-life, and negatively with tRNA adaptation indices. Similar results were obtained in mESCs, where the lag between IN and FP was even more pronounced with 35 - 38 min. We validated our measurements after stimulation of macrophages with lipopolysaccharide, where the lag between cytoplasmic and translated mRNA leads to a corresponding uncoupling between input and ribosome-protected fragments. Uncoupling is stronger for mRNAs with long ORFs or half-lives, a finding we also confirmed at the level of protein production by nascent chain proteomics. As a consequence of the lag in ribosome loading, ribosome density measurements are distorted when performed under conditions where mRNA levels are far from steady state expression, and transcriptional changes affect ribosome density in a passive way. This study uncovers an unexpected and considerable lag in ribosome loading, and provides guidelines for the interpretation of Ribo-Seq data taking passive effects on ribosome density into account.

2021-06-23 | GSE155233 | GEO

eIF1-eIF4G1 inhibitors uncover alternative translation activation of stress-response genes via enhanced ribosome loading and 5’UTR translation [Ribo-Seq and Ti-Seq]

Project description:The Ribo-seq and TI-seq analysis following i14G1s (eI1-eIF4G1 inhibitors) treatments uncover opposing roles of eIF1-eIF4G1 and eIF4E-eIF4G1 in scanning-dependent and independent translation. Furthermore, i14G1s inhibition of eIF4G1-eIF1 resulted in translation activation of ER/UPR stress-response genes via enhanced ribosome loading, elevated 5’UTR translation at near cognate AUGs, and unexpected concomitant upregulation of coding-region translation.

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Ribo-seq of Arabidopsis mTRAN double mutants

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Ribo-seq of HEK293T overexpressing WT and G240R GARS

Project description:Charcot-Marie-Tooth (CMT) disease can be caused by mutations in Aminoacyl-tRNA-Synthetases, including G240R mutation in Glycyl-tRNA-Synthetase (GARS). Ribo-seq generates snapshots of translating ribosomes on mRNA and therefore allows analysis of ribosome pausing mRNA. Here we performed Ribo-seq on lysates of HEK293T cells overexpressing GARS, WT or G240R, to dissect mechanism of CMT linked with translation. We found that GARS G240R causes pausing of ribosomes with glycine codons in A-site. The effect is specific for 21 nt ribosome-protected fragments, produced by ribosomes with empty A-sites, suggestive of the deficit of charged Glycyl-tRNA in GARS G240R-CMT.

2021-09-14 | E-MTAB-10342 | biostudies-arrayexpress

Ribo-seq in Escherichia coli MG1655 treated withTetracycline and Tylosin

Project description:Ribo-seq was performed on cells treated with Tetracycline and Tylosin to to investigate the effect on ribosome pausing across the transcriptome of Escherichia coli MG1655.

2022-10-15 | E-MTAB-12242 | biostudies-arrayexpress

Ribo-seq data from NIH 3T3 cells lacking eIF4G2

Project description:Eukaryotic translation initiation factor (eIF) 4G2 is a long-known homologue of the canonical eIF4G1. However, unlike the latter, it does not bind eIF4E or PABP, thus it remains unclear what and when brings eIF4G2 onto a ribosome. Here we report results of ribosome profiling performed in NIH 3T3 eIF4G2 (DAP5) -/- cells.

2020-10-19 | GSE158136 | GEO

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