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

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.