Periodic Reporting for period 1 - MOD-PROT (The role of tRNA processing and modifications in protein quality control.)
Reporting period: 2018-09-01 to 2020-08-31
This project aimed to explore the relationship between tRNA modifications and protein homeostasis (proteostasis). Our objective was to investigate whether modifications in the whole tRNA body (i.e. in addition to the anticodon) hampered translation speed and, if yes, how did it effect the cell’s proteostasis.
We focused on Mod5, a transfer RNA (tRNA) modifying enzyme required for the N6-isopentenylation of adenosine (i6A) at position 37 of cytoplasmatic and mitochondrial tRNATyr, tRNACys and tRNASer in bacteria, yeast, nematodes and humans. We found that yeast cells lacking this gene display codon-specific slow down at codons UCG (Ser), UAU (Tyr) and UGU (Cys). Transcriptomic and translatomic data revealed that the loss of the i6A modification increases mitochondrial translation while cytoplasmatic translation is decreased. Moreover, yeast mutants display a mild impairment of respiratory capacity. Finally, we found that MOD5 synthetically interacts with the mitochondrial ribosome quality control.
The project has been delayed by three months due to the Covid-19 related shutdown in Switzerland.
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.