Dissecting pre-tRNA splicing across scales: from molecules to cells

• Applicant: Dr. Simon Trowitzsch
• Subject Area: Biochemistry ; Structural Biology
• Term: since 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 428858078

Transfer RNAs (tRNAs) play a crucial role in protein biosynthesis by translating messenger RNA (mRNA) codons into amino acids at ribosomes. The maturation of precursor tRNAs (pre-tRNAs) involves the processing of their 5′ and 3′ ends, CCA addition, and removal of introns by splicing. Splicing is catalyzed by the tRNA splicing endonuclease complex (TSEN), while exon ligation is mediated by the tRNA splicing ligase complex (TSL). The human TSEN complex interacts with the RNA kinase CLP1, which paradoxically inhibits exon ligation. Mutations in TSEN and CLP1 have been linked to the neurodegenerative disorder pontocerebellar hypoplasia (PCH). My research group has made significant contributions to understanding the interactions between TSEN and pre-tRNAs in the context of PCH. Despite advancements in structural and biochemical studies, key questions regarding the molecular mechanisms of intron removal and exon ligation remain unresolved. This project aims to achieve a comprehensive understanding of pre-tRNA splicing through biochemical, structural, single-molecule, and cellular approaches. Specifically, we will analyze TSEN-CLP1 interactions, the kinetics of intron removal, the conformational dynamics of TSEN, the interaction between TSEN and TSL, as well as the spatiotemporal localization of pre-tRNA molecules and their splicing products in human cells. Using structural and biochemical studies, we will identify TSEN54-CLP1 binding motifs and investigate how PCH-associated mutations affect complex formation and pre-tRNA splicing. The kinetics of intron removal will be monitored using fluorescence spectroscopy and single-molecule imaging techniques with custom-designed pre-tRNA fluorophore/quencher probes. Structural snapshots of pre-tRNA-TSEN complexes will be obtained via time-resolved cryo-electron microscopy (cryo-EM). Molecular dynamics simulations will be employed to map these conformational transitions during catalysis. Through in-vitro reconstitution assays, we will investigate cooperativity between TSEN and TSL during the splicing process and provide structural insights into TSEN-TSL interactions using cryo-EM. Additionally, pre-tRNA processing pathways will be investigated in living cells using fluorescence-labeled pre-tRNA probes. Time-resolved confocal microscopy following microinjection will be employed to map the nuclear export and maturation routes of intron-containing pre-tRNAs, with findings validated through fractionation experiments. This multidisciplinary project integrates structural, biochemical, biophysical, and cellular approaches to comprehensively investigate pre-tRNA splicing. By uncovering novel regulatory pathways, this project will contribute substantially to our understanding of cellular RNA metabolism and its dysregulation in neurodegenerative diseases.

• DFG Programme: Research Grants