ABSTRACT
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 glycyl-tRNAGly available for translation and inhibit the first stage of elongation, the accommodation of glycyl-tRNA into the ribosomal A-site, which causes ribosomes to pause at glycine codons. Moreover, ribosome pausing activates a secondary repression mechanism at the level of translation initiation, by inducing the phosphorylation of the alpha subunit of eIF2 and the integrated stress response. Thus, CMT-GARS mutant triggers translational repression via two interconnected mechanisms, affecting both elongation and initiation of translation.
Subject(s)
Charcot-Marie-Tooth Disease/genetics , Glycine-tRNA Ligase/genetics , Peptide Chain Elongation, Translational/genetics , Peptide Chain Initiation, Translational/genetics , Ribosomes/metabolism , Cell Line , Eukaryotic Initiation Factor-2/metabolism , Gain of Function Mutation/genetics , Gene Expression/genetics , Glycine/genetics , HEK293 Cells , Humans , Phosphorylation , Protein Biosynthesis/genetics , RNA, Transfer, Gly/geneticsABSTRACT
The subcellular localization and translation of mRNAs are fundamental biological processes. In neurons, they underlie cell growth and synaptic plasticity, which serves as a foundation of learning and memory. Multiple approaches have been developed to separate neurons on subcellular compartments - cell bodies (soma) and cell extensions (axons and dendrites) - for further biochemical analyses. Here we describe neurite/soma separation approach in combination with RNA sequencing and proteomic analyses to identify localized and locally translated RNAs and proteins. This approach allows quantification of around 7000 of local proteins and the entire local transcriptome. It provides a powerful tool for investigation of the mechanisms underlying RNA localization and local translation in neurons.
