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bioRxiv · 10.1101/2022.09.03.506465

Structural basis of substrate recognition by human tRNA splicing endonuclease TSEN

Sekulovski, S.·Susac, L.·Stelzl, L. S.·Tampe, R.·Trowitzsch, S.
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Abstract

The heterotetrameric human transfer RNA (tRNA) splicing endonuclease (TSEN) catalyzes the excision of intronic sequences from precursor tRNAs (pre-tRNAs)1. Mutations in TSEN and its associated RNA kinase CLP1 are linked to the neurodegenerative disease pontocerebellar hypoplasia (PCH)2-8. The three-dimensional (3D) assembly of TSEN/CLP1, the mechanism of substrate recognition, and the molecular details of PCH-associated mutations are not fully understood. Here, we present cryo-electron microscopy structures of human TSEN with intron-containing pre-tRNATyrO_SCPLOWGTAC_SCPLOW and pre-tRNAArgO_SCPLOWTCTC_SCPLOW. TSEN exhibits broad structural homology to archaeal endonucleases9 but has evolved additional regulatory elements that are involved in handling and positioning substrate RNA. Essential catalytic residues of subunit TSEN34 are organized for the 3 splice site which emerges from a bulge-helix configuration. The triple-nucleotide bulge at the intron/3-exon boundary is stabilized by an arginine tweezer motif of TSEN2 and an interaction with the proximal minor groove of the helix. TSEN34 and TSEN54 define the 3 splice site by holding the tRNA body in place. TSEN54 adapts a bipartite fold with a flexible central region required for CLP1 binding. PCH-associated mutations are located far from pre-tRNA binding interfaces explaining their negative impact on structural integrity of TSEN without abrogating its catalytic activity in vitro10. Our work defines the molecular framework of pre-tRNA recognition and cleavage by TSEN and provides a structural basis to better understand PCH in the future.

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Sekulovski, S., Susac, L., Stelzl, L. S., Tampe, R., Trowitzsch, S.. 2022-09-03. Structural basis of substrate recognition by human tRNA splicing endonuclease TSEN. https://doi.org/10.1101/2022.09.03.506465

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