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Ribo-seq of HEK293T overexpressing WT and G240R GARS

ABSTRACT: 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.

INSTRUMENT(S): NextSeq 500

ORGANISM(S): Homo sapiens

SUBMITTER: Marina Chekulaeva

PROVIDER: E-MTAB-10342 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Charcot-Marie-Tooth mutation in glycyl-tRNA synthetase stalls ribosomes in a pre-accommodation state and activates integrated stress response.

Mendonsa Samantha S von Kuegelgen Nicolai N Bujanic Lucija L Chekulaeva Marina M

Nucleic acids research 20210901 17

Toxic gain-of-function mutations in aminoacyl-tRNA synthetases cause a degeneration of peripheral motor and sensory axons, known as Charcot-Marie-Tooth (CMT) disease. While these mutations do not disrupt overall aminoacylation activity, they interfere with translation via an unknown mechanism. Here, we dissect the mechanism of function of CMT mutant glycyl-tRNA synthetase (CMT-GARS), using high-resolution ribosome profiling and reporter assays. We find that CMT-GARS mutants deplete the pool of g ...[more]

PMID: 34403468

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