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The role of tRNA processing and modifications in protein quality control.

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

Life is possible because cells have the inherent capacity to translate their genetic information into functional proteins. This process is called translation and within this complex and tightly regulated process, transfer RNAs (tRNAs) take a fundamental role by bringing the correct amino acid to the growing nascent peptide. tRNAs are highly modified molecules because they undergo several posttranscriptional chemical modifications. However, the relevance of many of those chemical modifications in tRNA maturation and, more importantly, during translation remains to be understood. Previously, the host group discovered that cells lacking certain tRNA modifications in the anticodon induce translational slow down, a defect that triggers widespread protein aggregation in yeast and nematodes. This was the first evidence linking tRNA metabolism to protein quality control and established tRNA metabolism as a novel layer in the regulation of protein homeostasis.
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.
In this project, we sought to elucidate how tRNA processing and modifications regulate protein quality control, by integrating genetic, biochemical, transcriptomic and translatomic approaches. We set out to characterize Mod5 by asking what consequences arise upon loss of the i6A modification during translation and proteome integrity. First, we confirmed by quantitative mass spectrometry that the loss of Mod5 abolished the i6A modification in yeast cells. Second, our polysome profiles showed that the loss of this tRNA modification resulted in a reduction of global translation. We next performed ribosome profiling to obtain insights into translation dynamics at codon resolution. We found codon-specific ribosome pausing of one hypomodified codon, UCG, which is read by tSer(CGA) while the other hypomodified codons remained largely unaffected. Interestingly, we identified two additional codons that were slowed down, UAU (Tyr) and UGU (Cys), which do not have cognate tRNAs. Our results indicate that these codons require the i6A modification on the non-cognate tRNATyr(GUA) and tRNACys(GCA) in order to be read during translation. Under steady-state conditions, we also found that the deletion of Mod5 is associated with compromised mitochondrial function. Curiously, the human homologue of Mod5 (TRIT1) has been implicated in mitochondriopathies and our findings could uncover basic cellular processes that link translation dynamics to mitochondria in health and disease. We will continue actively disseminating our (yet unpublished) findings in (inter)national conferences related to RNA and mitochondria biology, and we expect that we will be able to publish this work in a high-impact peer-reviewed scientific journal like eLife or Journal of Cell Biology.
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.