Structural Basis of Pre-tRNA Intron Removal by Human tRNA Splicing Endonuclease

Removal of intron from precursor-tRNA (pre-tRNA) is essential in all three kingdoms of life. In human, this process is mediated by the tRNA splicing endonuclease (TSEN) that consists of four subunits: TSEN2, TSEN15, TSEN34, and TSEN54. Here we report the cryo-EM structures of human TSEN bound to full-length pre-tRNA in the pre-catalytic and post-catalytic states at average resolutions of 2.94-Å and 2.88-Å, respectively. Human TSEN features an extended surface groove that holds the L-shaped pre-tRNA. The mature domain of pre-tRNA is recognized by conserved structural elements of TSEN34, TSEN54 and TSEN2. Such recognition orients the anticodon stem of pre-tRNA and places the 3’-splice site and 5’-splice site into the catalytic centers of TSEN34 and TSEN2, respectively. The bulk of the intron sequences make no direct interaction with TSEN, explaining why pre-tRNAs of varying introns can be accommodated and cleaved. Our structures reveal the molecular ruler mechanism of pre-tRNA cleavage by TSEN.