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Methods comparison for ribosome profiling

ABSTRACT: Assessment of the effecacy of direction extraction of ribosome-protected mRNA fragments for ribosome profiling

ORGANISM(S): Homo sapiens

PROVIDER: GSE68008 | GEO | 2015/07/01

SECONDARY ACCESSION(S): PRJNA281481

REPOSITORIES: GEO

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Methods comparison for ribosome profiling

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Project description:We report the changes in ribosome protected fragments as measured by ribosome profiling in cells with or without depletion of tRNA-Tyr-GUA

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Project description:Ribosome profiling and RNAseq data on human BJ fibroblasts and cybrid cells using an adapted ribosome profiling protocol to improve detection of mitochondrial ribosome protected fragments 51-base length single read ribosome profiling data on human fibroblasts and cybrid cells using an adapted ribosome profiling protocol; and 51-base length single read RNAseq on polyA enriched RNA

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Project description:Ribosomes undergo substantial conformational changes during translation elongation to accommodate incoming aminoacyl-tRNAs and translocate along the mRNA template. We used multiple elongation inhibitors and chemical probing to define ribosome conformational states corresponding to different sized ribosome-protected mRNA fragments (RPFs) generated by ribosome profiling. We show using various genetic and environmental perturbations that the previously identified 20-22 nucleotide (nt) RPFs correspond predominantly to ribosomes in a pre-accommodation state with an open 40S ribosomal A site while the classical 27-29 nt fragments correspond to ribosomes in a pre-translocation state with an occupied 40S ribosomal A site. These distinct ribosome conformational states revealed by ribosome profiling are seen in all eukaryotes tested including fungi, worms and mammals. This high-resolution ribosome profiling approach reveals the anticipated Rck2-dependent inhibition of translocation through eEF2 phosphorylation during hyperosmotic stress. These same approaches reveal a strong translation elongation arrest during oxidative stress where the ribosomes are trapped in a pre-translocation state, but in this case the translational arrest is independent of Rck2-driven eEF2 phosphorylation. These results provide new insights and approaches for defining the molecular events that impact translation elongation throughout biology.

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