Enzymology of the metazoan tRNA ligase complex: a lifetime in cycles.

This review highlights the emerging biochemistry and biology of the metazoan tRNA-ligase complex (tRNA-LC). We begin with an overview of the cleavage-ligation pathways dependent on the tRNA-LC, epitomised by the essential process of pre-tRNA splicing, but also critical for the unfolded protein response and the struggle against transposons. The catalytic core of the tRNA-LC is the non-conventional RNA ligase RTCB, found in all domains of life. We start from the universal principles of its catalytic cycle, entailing GTP-dependent ligation of RNA molecules with 2’,3’-cyclic phosphate and 5’-hydroxyl ends. We then focus on the new findings that govern trafficking, protection, regulation and degradation of the tRNA-LC. These new modalities arise from an expanded set of subunits that to a large extent specifically associate with RTCB in Eukarya. We present how co-purification and co-evolutionary analyses converged to guide sequential discoveries of these proteins and illuminated their biochemical roles. We detail how the choice of paralogue of the auxiliary subunit FAM98 and the recruitment of Ashwin determine RTCB localisation, discriminating between its cytoplasmic and nuclear roles. We then pay particular attention to discoveries emerging from the latest structural works. These include the Archease-mediated mechanism of metazoan RTCB guanylylation, and PYROXD1-mediated protection of the tRNA-LC in the “resting state” from oxidative inactivation. We illustrate how metal ions play critical roles in both of these processes, alongside their direct roles in catalysing RNA ligation and, potentially, in the degradation of the tRNA-LC through a novel and still mysterious mechanism. We place particular emphasis on the subunits with contested functions, and on the interplay between their role in the tRNA-LC and their other cellular tasks. Along the way, we highlight the links between the subunits of the tRNA-LC, in particular their pathogenic variants or their mis-localisation, and human diseases including neurodegeneration and myopathies. The increasing understanding of the tRNA-LC, including its expanded set of subunits, illustrates how branching out from a single enzyme uncovered new biology ranging from the previously unknown congenital myopathies to novel proteasome targeting pathways.