Structural and biochemical characterization of the 3'-5' tRNA splicing ligases

J Biol Chem. 2025 May;301(5):108506. doi: 10.1016/j.jbc.2025.108506. Epub 2025 Apr 10.

Abstract

In archaea and metazoa, tRNA exons are ligated by the RNA ligases RtcB and RTCB, respectively. The metazoan RTCB forms a stable complex with four additional subunits, DDX1, FAM98B, CGI99, and ASHWIN. The role and assembly of these four components remain elusive. Furthermore, we lack structural information of how RNA substrates are recognized by 3'-5' tRNA ligases. Here, we use thiol-based chemical crosslinking to confirm the involvement of specific residues of RtcB in RNA binding, and we present a cryo-EM structure of the purified five-subunit Danio rerio tRNA ligase complex. The structure implies that the DDX1 helicase module is mobile and can modulate the activity of RTCB. Taken together, the presented results enhance our mechanistic understanding of RNA binding by 3'-5' tRNA splicing ligases and architecture of the metazoan tRNA ligase complex.

Keywords: RNA splicing; RNA-binding protein; RTCB; pre-tRNA splicing; protein complex; protein structure; protein–nucleic acid interaction; structural biology; tRNA; tRNA ligase complex.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cryoelectron Microscopy
  • DEAD-box RNA Helicases / chemistry
  • DEAD-box RNA Helicases / genetics
  • DEAD-box RNA Helicases / metabolism
  • Models, Molecular
  • RNA Ligase (ATP)* / chemistry
  • RNA Ligase (ATP)* / genetics
  • RNA Ligase (ATP)* / metabolism
  • RNA Splicing*
  • RNA, Transfer* / chemistry
  • RNA, Transfer* / genetics
  • RNA, Transfer* / metabolism
  • Zebrafish / metabolism
  • Zebrafish Proteins* / chemistry
  • Zebrafish Proteins* / genetics
  • Zebrafish Proteins* / metabolism

Substances

  • RNA Ligase (ATP)
  • DEAD-box RNA Helicases
  • RNA, Transfer
  • Zebrafish Proteins

Associated data

  • PDB/9I8V