Project B19 – Structural and mechanistic basis of pre-tRNA processing by the human tRNA splicing endonuclease in health and neurodegenerative disease

Working on this project:

Simon Trowitzsch & Team

Introns of human transfer RNA precursors (pre-tRNAs) are excised by the tRNA splicing endonuclease TSEN in complex with the RNA kinase CLP1. Mutations in TSEN/CLP1 occur in patients with pontocerebellar hypoplasia (PCH), however, their role in the disease is unclear. In this project we will address fundamental questions on pre-tRNA processing by three major objectives. By solving high-resolution structures of the human endonuclease, we will provide a structural framework for understanding the molecular mechanism of pre-tRNA splicing, explain differences between archaeal and eukaryotic systems, provide evidence for the role of CLP1, reveal the structure of an intron-containing pre-tRNA molecule, and explain the impact of disease mutations. We will track pre-tRNA splicing in single cells at sub-cellular resolution using tailored pre-tRNA fluorophore/quencher probes that report on localization and splice status. We will use this tRNA reporter system in a cellular disease model to study consequences of mutations on localization of TSEN components, tRNA and splicing. Furthermore, the pre-tRNA probes will allow us to deduce real-time kinetics of intron excision for wild type and mutant endonuclease complexes in vitro. Ultimately, we will identify novel target RNAs of the human pre-tRNA endonuclease by UV cross-linking immunoprecipitation-high-throughput sequencing (CLIP-seq). Moonlighting activities on messenger and ribosomal RNAs have been identified for the yeast tRNA endonuclease. Our data will for the first time reveal direct involvement of the human pre-tRNA endonuclease in cellular processes distinct from pre-tRNA splicing possibly enforced by disease-causing mutations.