NCN OPUS 26 (LAP) 2024 – 2027

The role of tRNA thiolation in human disease

Glatt-lab

The background

All living cells require ribosomes and tRNA molecules to decode and translate the genomic information stored in mRNAs into correctly assembled nascent polypeptide chains. The presence of unique chemical modifications in anticodons of tRNAs not only adds additional heterogeneity but also complexity to the process of codon-anticodon mediated decoding and hence regulation of de novo protein synthesis. These cellular mechanisms are clinically highly relevant as genomic mutations in various RNA modifying enzymes have been directly linked to the onset of severe human pathophysiological conditions, which include a spectrum of rare syndromes, neurodegenerative diseases, obesity, deafness and cancer. The URM1/MOCS3 pathway is responsible for catalyzing the s2-thiouridine formation on wobble base uridines in four eukaryotic tRNA species. In detail, tRNALysUUU, tRNAGluUUC, tRNAGlnUUG and tRNAArgUCU that have undergone a preceding modification at their C5 position by the Elongator complex, receive a sulfur (thiol) at their C2 position, resulting in an mcm5s2U34 modified wobble base. Recent studies have found that this modification is crucial for proteome homeostasis by directly influencing the speed of translating ribosomes. During the final step of the enzymatic cascade, sulfur is passed onto the wobble uridine via the CTU1/CTU2 thioltransferase complex, which binds tRNAs and is required for the last step of the modification reaction. The mechanistic details of the final sulfur transfer to U34 remain poorly characterized and we lack a fundamental understanding of the reaction carried out by the human CTU1/CTU2 complex. Pathogenic CTU2 variants are causally linked to the development of the human DREAM-PL syndrome. This clinical link underscores the importance of characterizing the human thiolation cascade. Patients with DREAM-PL show dramatic developmental defects including dysmorphic facies, renal agenesis, ambiguous genitalia, microcephaly, polydactyly and lissencephaly. Many of these clinical features are shared with ciliopathies. However, the consequences of impaired tRNA thiolation at the cellular level, and the pathomechanism(s) underlying this devastating human syndrome remain unknown.

Research interest

The proposed project aims to go significantly beyond the current state-of-the-art by addressing the following two fundamental questions:
• How do changes in tRNA thiolation levels cause a severe human disease?
• How does the human CTU1/CTU2 complex catalyze tRNA thiolation?

The project

The strength of this proposal stems from the highly complementary knowledge of the collaboration partners in Krakow and Vienna, our long-standing expertise in the field of tRNA modifications, a wide range of established experimental tools nd the direct access to state-of-the-art research facilities. We will apply a combination of in vivo and in vitro approaches to obtain a comprehensive molecular understanding of the human CTU1/CTU2 complex, to characterize the specific consequences of a lack of thiolation in human cells and to define the role of specific patient-derived mutations linked to DREAM-PL syndrome. The project will build on the extensive know-how and toolboxes that we have developed throughout our previous work on the fungal thiolation pathway and initial characterization of DREAM-PL patient cells.
Our long-standing competence in field has resulted in excellent collaborations and a large network of partners in Switzerland, Germany, USA, Belgium, Poland and Austria that are accessible to complement our own expertise.

The significance

The expected results from the proposed research project will provide deep molecular insights into the yet poorly characterized pathophysiological consequences of defective tRNA thiolation. Together with the structural insights of the human thiolation machinery our results will substantiate our understanding on the interplay of tRNA thiolation, protein homeostasis and its contribution to severe human pathologies. The project will contribute to identifying diagnostic biomarkers and potential intervention points for future targeted therapies against DREAM-PL syndrome and related diseases.