RtcB enzymes are RNA ligases that play essential roles in tRNA splicing, unfolded protein response, and RNA repair. In metazoa, RtcB functions as part of a five-subunit tRNA ligase complex (tRNA-LC) along with Ddx1, Cgi-99, Fam98B and Ashwin. The human tRNA-LC or its individual subunits have been implicated in additional cellular processes including microRNA maturation, viral replication, DNA double-strand break repair and mRNA transport. Here we present a biochemical analysis of the inter-subunit interactions within the human tRNA-LC along with crystal structures of the catalytic subunit RTCB and the N-terminal domain of CGI-99. We show that the core of the human tRNA-LC is assembled from RTCB and the C-terminal alpha-helical regions of DDX1, CGI-99, and FAM98B, all of which are required for complex integrity. The N-terminal domain of CGI-99 displays structural homology to calponin-homology domains, and CGI-99 and FAM98B associate via their N-terminal domains to form a stable subcomplex. The crystal structure of GMP-bound RTCB reveals divalent metal coordination geometry in the active site, providing insights into its catalytic mechanism. Collectively, these findings shed light on the molecular architecture and mechanism of the human tRNA ligase complex, and provide a structural framework for understanding its functions in cellular RNA metabolism.

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RNA molecules are frequently modified with a terminal 2′,3′-cyclic phosphate group as a result of endonuclease cleavage, exonuclease trimming, or de novo synthesis. During pre-transfer RNA (tRNA) and unconventional messenger RNA (mRNA) splicing, 2′,3′-cyclic phosphates are substrates of the tRNA ligase complex, and their removal is critical for recycling of tRNAs upon ribosome stalling. We identified the predicted deadenylase angel homolog 2 (ANGEL2) as a human phosphatase that converts 2′,3′-cyclic phosphates into 2′,3′-OH nucleotides. We analyzed ANGEL2’s substrate preference, structure, and reaction mechanism. Perturbing ANGEL2 expression affected the efficiency of pre-tRNA processing, X-box–binding protein 1 (XBP1) mRNA splicing during the unfolded protein response, and tRNA nucleotidyltransferase 1 (TRNT1)–mediated CCA addition onto tRNAs. Our results indicate that ANGEL2 is involved in RNA pathways that rely on the ligation or hydrolysis of 2′,3′-cyclic phosphates.

Non-templated 3′-uridylation of RNAs has emerged as an important mechanism for regulating the processing, stability and biological function of eukaryotic transcripts. In Drosophila, oligouridine tailing by the terminal uridylyl transferase (TUTase) Tailor of numerous RNAs induces their degradation by the exonuclease Dis3L2, which serves functional roles in RNA surveillance and mirtron RNA biogenesis. Tailor preferentially uridylates RNAs terminating in guanosine or uridine nucleotides but the structural basis underpinning its RNA substrate selectivity is unknown. Here, we report crystal structures of Tailor bound to a donor substrate analog or mono- and oligouridylated RNA products. These structures reveal specific amino acid residues involved in donor and acceptor substrate recognition, and complementary biochemical assays confirm the critical role of an active site arginine in conferring selectivity toward 3′-guanosine terminated RNAs. Notably, conservation of these active site features suggests that other eukaryotic TUTases, including mammalian TUT4 and TUT7, might exhibit similar, hitherto unknown, substrate selectivity. Together, these studies provide critical insights into the specificity of 3′-uridylation in eukaryotic post-transcriptional gene regulation.

RTCB is the catalytic subunit of the metazoan tRNA ligase complex (tRNA-LC) that plays essential roles in tRNA biogenesis and unfolded protein response. The catalytic center of RTCB contains a conserved cysteine that is susceptible to metal ion-induced oxidative inactivation. The flavin-containing oxidoreductase PYROXD1 preserves the activity of mammalian tRNA-LC in a NAD(P)H-dependent manner, but its protective mechanism remains elusive. Here we report a cryo-EM structure of human RTCB in complex with PYROXD1, revealing that PYROXD1 directly interacts with the catalytic center of RTCB through its C-terminal tail. NAD(P)H binding and FAD reduction allosterically control PYROXD1 activity and RTCB recruitment and PYROXD1, while PYROXD1 reoxidation enables timed release of RTCB. PYROXD1 interaction is mutually exclusive with Archease-mediated RTCB guanylylation, and guanylylated RTCB is intrinsically protected from oxidative inactivation. Together, these findings provide a mechanistic framework for the protective function of PYROXD1 that maintains the activity of tRNA-LC under aerobic conditions.