As there are at least two proposed mechanisms of re-ligating “half” tRNAs in mammals we set out to uncover the requirement for CLP1 in human cells. To test for altered cell viability of CLP1 mutant cells, we performed growth analysis of fibroblasts and iNeurons under basal conditions, but detected no differences (Figure S5A-B). We next tested whether overexpression of pre-tRNAs compared with mature tRNAs were toxic in patient iNeurons in culture, since iNeurons displayed an accumulation of these pre-tRNAs. Using a lentiviral delivery system expressing Chr14:tRNA19-TyrGTA or Chr19:tRNA10-IleTAT pre- or processed tRNA (both pre isoforms accumulated by Northern analysis in patient iNeurons), we found no difference in viability between patient and control iNeurons 24h after transduction (Figure S5C). We conclude that the patient iNeurons do not show a dose-dependent toxicity to pre-tRNAs under these conditions.
