Intron excision from precursor tRNA molecules in mammalian cells requires ATP hydrolysis and phosphorylation of tRNA-splicing endonuclease components

The process of tRNA splicing entails removal of an intron by TSEN (tRNA–splicing endonuclease) and ligation of the resulting exon halves to generate functional tRNAs. In mammalian cells, the RNA kinase CLP1 (cleavage and polyadenylation factor I subunit) associates with TSEN and phosphorylates the 3′ exon at the 5′ end in vitro, suggesting a role for CLP1 in tRNA splicing. Interestingly, recent data suggest that the ATP-binding and/or hydrolysis capacity of CLP1 is required to enhance pre-tRNA cleavage. In vivo, the lack of CLP1 kinase activity leads to progressive motor neuron loss and accumulation of novel 5′ leader–5′ exon tRNA fragments. We have extended the investigation of the biochemical requirements in pre-tRNA splicing and found that β–γ-hydrolysable ATP is crucial for the productive generation of exon halves. In addition, we provide evidence that phosphorylation of the TSEN complex components supports efficient pre-tRNA cleavage. Taken together, our data improve the mechanistic understanding of mammalian pre-tRNA processing and its regulation.