Structural basis of pre-tRNA intron removal by human tRNA splicing endonuclease

Removal of the intron from precursor-tRNA (pre-tRNA) is essential in all three kingdoms of life. In humans, this process is mediated by the tRNA splicing endonuclease (TSEN) comprising 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 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 makes 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. [Display omitted] •Structures of human TSEN/pre-tRNA complex in pre-catalytic and post-catalytic states•Recognition of the full-length pre-tRNA by human TSEN•Recognition and cleavage of the 3′-splice site and 5′-splice site•Structural insights into the “molecular ruler” mechanism Zhang et al. report the cryo-EM structures of human TSEN bound to full-length pre-tRNA in the pre-catalytic and post-catalytic states. The structures show how TSEN recognizes the pre-tRNA and orients the 3′-splice site and 5′-splice site into its cleavage sites. The structural and biochemical work explains the molecular ruler mechanism of pre-tRNA intron removal by human TSEN.