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.
X-ray diffraction data and atomic models have been deposited in the Protein Data Bank under accession codes 7P3A (CGI-99 N-terminal domain) and 7P3B (RTCB in complex with GMP and Co(II)).The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the dataset identifier PXD025662
N-terminal domain of CGI-99Protein Data Bank, 7P3A.
Human RNA ligase RTCB in complex with GMP and Co(II)Protein Data Bank, 7P3B.
tRNA-LC cross-linking/mass spectrometry dataProteomeXchange, PXD025662.
- Alena Kroupova
- Alena Kroupova
- Fabian Ackle
- Franziska M Boneberg
- Alexander Leitner
- Martin Jinek
- Igor Asanović
- Stefan Weitzer
- Javier Martinez
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
- Timothy W Nilsen, Case Western Reserve University, United States
© 2021, Kroupova et al.
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