This project has achieved a large fraction of its original objectives and milestones. However, corrective action had to be taken. Therefore, we will continue this project beyond the duration of the fellowship.
First, we have improved our ribosome profiling protocol, yielding excellent frame information quality and sequencing depth of mapped reads of our yeast mutants. In collaboration with a PhD student in the host lab (Jie Wu), we have also optimized our own sequencing data analysis package. These improvements have allowed us to uncover several aspects of translation dynamics in our yeast deletion mutants for tRNA modifications and ribosome quality control, including codon-specific ribosomal pausing and dicodon analysis.
Second, our work on the tRNA modification enzyme Mod5 revealed a unique, dual reaction of the cell’s loss of the i6A modification: on the one hand, the modification is essential for non-cognate tRNAs (for tyrosine and cysteine) to properly decode mRNA while, on the other hand, the modification is essential albeit for one single cognate tRNA (for serine). This finding is correlated with changes in the mitochondria of MOD5 yeast deletion mutants, in particular the increase of mitochondrial translation in comparison to cytoplasmatic translation and a mild impairment of respiratory capacity. Interestingly, mutations in the human homologue of Mod5 (TRIT1) causes inherited mitochondrial diseases, and only two studies have been published so far (Kernohan et al, 2017; Yarham et al, 2014). The mechanisms, by which mutations in TRIT1 cause disease have remained elusive. Our findings will provide insights into the cellular mechanisms, by which defects in translation lead to compromised mitochondrial function in humans. We are currently complementing our genetic, transcriptomic and translatomic data with biochemical experiments to uncover how loss of Mod5 impairs mitochondria health.
