To test for defective tRNA splicing, we generated cDNA from fibroblasts and iNeurons, then analyzed expression of these pre-tRNAs using qRT-PCR to test for differences between affected and unaffected. Normalizing to the unaffected in fibroblasts, in the half of pre-tRNAs where a difference was noted, the differences were roughly equally distributed into those where the pre-tRNA was higher in the affected and those where it was lower in the affected (Figure 4B, S4B). In iNeurons, however, where a difference in pre-tRNAs level was noted, the majority (6 of 8) showed an accumulation of pre-tRNA in affected cells compared with unaffected (Figure 4C, S4C). We conclude that there is an accumulation of some pre-tRNAs in CLP1 patient cells.
