We transfected control and CLP1R140H mutant fibroblasts with RNA oligonucleotides corresponding to the most abundantly expressed Tyr tRNA intermediates, either in the absence or presence of HP and evaluated cell viability after 24h (Figure 5B-C). Control fibroblasts and neural progenitor cells showed little toxicity in response to the tRNA intermediates, either in the absence or presence of HP (Figure S5H-I, K). Co-treatment with the 5’-exon containing the terminal 2’-3’-cyclic phosphodiester did not have a notable effects on survival (Figure S5H). Most strikingly, the unphosphorylated 3’-exon was the most lethal of the tRNA halves, particularly in the presence of HP (Figure 5B, S5I). Remarkably, the 5’-phosphorylated 3’-exon did not cause toxicity and in fact significantly improved viability in the presence of HP (Figure 5C, S5J). These results are consistent with the model whereby the loss of the phosphorylated-3’-exon required for HSPC117-independent tRNA ligation (and therefore production of mature tRNAs) or the toxic hydroxyl-3’-exon (incapable of further processing in mutant cells), may contribute significantly to the disease phenotype in patients.
