Biology subjects

Boneberg, F. M.

Publications and source records attributed to Boneberg, F. M..

3 recordsLinked to original sources

bioRxiv2023

Mechanistic basis for oxidative stress protection of the human tRNA ligase complex by the oxidoreductase PYROXD1

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.

biochemistry↗

bioRxiv2022

Fip1 is a multivalent interaction scaffold for processing factors in human mRNA 3' end biogenesis

3 end formation of most eukaryotic mRNAs is dependent on the assembly of a [~]1.5 megadalton multiprotein complex, that catalyzes the coupled reaction of pre-mRNA cleavage and polyadenylation. In mammals, the cleavage and polyadenylation specificity factor (CPSF) constitutes the core of the 3 end processing machinery onto which the remaining factors, including cleavage stimulation factor (CstF) and poly(A) polymerase (PAP), assemble. These interactions are mediated by Fip1, a CPSF subunit characterized by high degree of intrinsic disorder. Here, we report two crystal structures revealing the interactions of human Fip1 (hFip1) with CPSF30 and CstF77. We demonstrate that CPSF contains two copies of hFip1, each binding to the zinc finger (ZF) domains 4 and 5 of CPSF30. Using polyadenylation assays we show that the two hFip1 copies are functionally redundant in recruiting one copy of PAP, thereby increasing the processivity of RNA polyadenylation. We further show that the interaction between hFip1 and CstF77 is mediated via a short motif in the N-terminal "acidic" region of hFip1. In turn, CstF77 competitively inhibits CPSF-dependent PAP recruitment and 3 polyadenylation. Taken together, these results provide a structural basis for the multivalent scaffolding and regulatory functions of hFip1 in 3 end processing.

biochemistry↗

bioRxiv2021

Molecular architecture of the human tRNA ligase complex

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.

biochemistry↗