Structural framework for the assembly of the human tRNA ligase complex

In human cells, a subset of tRNA-encoding genes contain introns. These are removed by a spliceosome-independent pathway in which the tRNA splicing endonuclease complex catalyzes intron excision. The resulting exons are subsequently ligated by the tRNA-ligase complex (tRNA-LC), comprising Ashwin, CGI-99, FAM98B, DDX1, and RTCB/HSPC117. The molecular architecture and functions of its non-catalytic subunits remain poorly understood. Using cryo-EM, we determined an atomic-resolution structure of human tRNA-LC. CGI-99, DDX1, and FAM98B form an α-helical bundle that contacts RTCB opposite its active site and anchors DDX1 via its C-terminal helix. FAM98B and CGI-99 form an extensively co-folded heterodimer that clamps Ashwin in a pincer-like structure. Structure-based mutagenesis supports the architecture of the complex. We further show that FAM98A and FAM98C assemble distinct RTCB-containing complexes lacking Ashwin, suggesting specialized cellular functions. Our results provide insights into the molecular assembly of the tRNA ligase complex, highlighting its functions in tRNA biogenesis and beyond.