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

点击翻译按钮获取中文摘要

Transfer RNAs (tRNAs) are among the most ubiquitous molecules in cells from all three domains of life and central to decoding information from messenger RNAs to proteins on translating ribosomes. Beyond their canonical role during protein biosynthesis, tRNAs also perform additional functions as signaling molecules in the regulation of numerous metabolic and cellular processes, as stress sensors and in tRNA-dependent biosynthetic pathways. tRNA fragments have been identified as novel species of small non-coding RNAs contributing to translational control, gene regulation and silencing, and progressive motor neuron loss. tRNAs are encoded as precursor molecules, which undergo a plethora of modifications, including removal of intronic sequences. In humans, introns are cleaved by the heterotetrameric tRNA splicing endonuclease (TSEN), which associates with the RNA kinase CLP1. Mutations in TSEN subunits or CLP1 lead to the development of severe neurological disorders. How these mutations engage in the development of the disease is totally unclear. Previous studies revealed first functional and structural aspects of the human pre-tRNA splicing endonuclease, but detailed insights into the assembly of the endonuclease subunits and substrate recognition remain elusive due to the lack of high-resolution structures. General principles of pre-tRNA recognition and cleavage by TSEN were only deduced from biochemical studies. The role of CLP1 in the process of pre-tRNA splicing remains enigmatic. I 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.