The ligation steps of tRNA splicing in yeast and vertebrate cells have been thought to proceed by fundamentally different mechanisms. Ligation in yeast cells occurs by incorporation of an exogenous phosphate from ATP into the splice junction, with concomitant formation of a 2' phosphate at the 5' junction nucleotide. This phosphate is removed in a subsequent step which, in vitro, is catalyzed by an NAD-dependent dephosphorylating activity. In contrast, tRNA ligation in vertebrates has been reported to occur without incorporation of exogenous phosphate or formation of a 2' phosphate. We demonstrate in this study the existence of a yeast tRNA ligase-like activity in HeLa cells. Furthermore, in extracts from these cells, the entire yeastlike tRNA splicing machinery is intact, including that for cleavage, ligation, and removal of the 2' phosphate in an NAD-dependent fashion to give mature tRNA. These results argue that the mechanism of tRNA splicing is conserved among eukaryotes.
Yeast tRNA-splicing endonuclease cleaves precursor tRNA in a random pathway.
