Reconstitution of Human tRNA Intron Cleavage Leads to an Updated Model of tRNA Splicing

Abstract

Transfer RNAs (tRNAs) are critical for translation. tRNAs require extensive post-transcriptional maturation, including the removal of an intron for a subset of tRNAs. tRNA splicing is a multi-step process that involves the cleavage of introns and the ligation of tRNA exons back together. In eukaryotes, tRNA introns are removed by the heterotetrametric tRNA splicing endonuclease (TSEN) complex. In metazoans, ligation is carried out by the tRNA ligase, RTCB, which also ligates introns into tRNA intronic circular RNAs (tricRNAs). Furthermore, the metazoan TSEN complex associates with an accessory protein 5’-hydroxyl polyribonucleotide kinase CLP1. Until recently, the function of CLP1 in tRNA splicing was thought to be in supporting TSEN function/stability as mutations in CLP1 and the TSEN proteins are known to lead to a family of neurological and neurodegenerative diseases, pontocerebellar hypoplasia (PCH). Despite these connections, non-metazoan eukaryotes do not utilize CLP1 in tRNA splicing. To address the role of CLP1 in TSEN function and tRNA splicing, we developed the first system for the production of recombinant active TSEN complex from E. coli and complemented this approach with immunoprecipitations from HEK cells. We showed for the first time that CLP1 is not required for the TSEN complex to remove tRNA introns, uniting how eukaryotes cleave tRNA introns. We then utilized a reporter tRNA to monitor tRNA splicing in cells. We overexpressed CLP1 and kinase-dead CLP1 in HEK cells and monitored splicing of our reporter tRNA. We found that kinase-active but not kinase-dead CLP1 reduced the production of tricRNAs, suggesting that CLP1 might negatively regulate the ligation step of tRNA splicing. In parallel, we knocked down CLP1's homolog, cbc, in Drosophila cells and looked at the maturation of a Drosophila-specific tRNA reporter. We found that depletion of cbc led to a significant increase in both mature tRNA as well as tricRNAs for our reporter construct. Together, our data support a new model of tRNA splicing in which CLP1 is a negative regulator of tRNA and tricRNAs maturation. Hayne, et al. Reconstitution of the human tRNA splicing endonuclease complex: insight into the regulation of pre-tRNA cleavage (2020), Nucleic Acids Research.