Structure of human RNA polymerase III

Abstract

In eukaryotes, RNA Polymerase (Pol) III is specialized for the transcription of tRNAs and other short, untranslated RNAs. Pol III is a determinant of cellular growth and lifespan across eukaryotes. Upregulation of Pol III transcription is observed in cancer and causative Pol III mutations have been described in neurodevelopmental disorders and hypersensitivity to viral infection. Here, we report a cryo-EM reconstruction at 4.0 Å of human Pol III, allowing mapping and rationalization of reported genetic mutations. Mutations causing neurodevelopmental defects cluster in hotspots affecting Pol III stability and/or biogenesis, whereas mutations affecting viral sensing are located in proximity to DNA binding regions, suggesting an impairment of Pol III cytosolic viral DNA-sensing. Integrating x-ray crystallography and SAXS, we also describe the structure of the higher eukaryote specific RPC5 C-terminal extension. Surprisingly, experiments in living cells highlight a role for this module in the assembly and stability of human Pol III.

POLR3-Related Leukodystrophy: Exploring Potential Therapeutic Approaches

“…Although the cellular pathophysiological mechanisms associated with POLR3-HLD have yet to be uncovered, studies have shown that mutations in POLR3 subunits can cause disruptions on several molecular levels. For example, mutational mapping onto specific protein domains suggests association with specific mechanisms of dysfunction, including modification of the catalytic cleft structure, impaired POLR3 complex assembly, perturbed interactions between subunits, and interference within POLR3 complex binding to DNA (Bernard et al, 2011 ; Tétreault et al, 2011 ; Girbig et al, 2020 ; Ramsay et al, 2020 ). Additionally, protein localization studies have shown that disease-causing POLR1C variants can alter assembly and nuclear import exclusively of POLR3, resulting in a lack in binding to POLR3 target genes (Thiffault et al, 2015 ).…”

Coordination of transcription and processing of tRNA

“…Moreover, knockdown of RNase P leads to a decrease in the binding of RPC6 and RPC7 to 5S rRNA and tRNA gene loci [73]. This finding highlights the necessity for an intact RNase P in formation of the initiation subcomplex, which is positioned close to the start point of transcription [52,66,67,68,100].…”

“…Pol III has seventeen protein subunits [60][61][62], three of which form a specific subcomplex, RPC3-RPC6-RPC7, implicated in promoter-dependent initiation and elongation [52,62,63,64,65,66,67]. The cryo-EM structure of human Pol III reveals several species-specific modules that mediate activation and repression of transcription [64,65,68]. The core transcription factors TFIIIA, TFIIIB, TFIIIC, and SNAPc assemble diverse initiation complexes of Pol III on target genes with various types of promoters and auxiliary cis-acting sequences [51,53,54,61,63,69,70].…”

“…Even though these enzymatic activities are likely to correspond to those of RNase P, RNase Z, and tRNA splicing complex, further work that includes genetic, reverse genetic, and rescue experiments is needed to conclude the findings (Ramanathan A. and Mani D., in preparation). Nonetheless, preliminary results uncover that purified type II initiation complexes have high molecular weights of 2-3 9 10 6 (Ramanathan A., and Mani D., unpublished data), large enough to accommodate RNase P (~500 kDa), RNase Z (~85 kDa), tRNA endonuclease complex (~190 kDa), and tRNA ligase complex (~300 kDa) [108], along with Pol III (~700 kDa) [68], TFIIIB, and TFIIIC and other auxiliary transcription factors. Mass spectrometry analyses confirm the presence of numerous tRNA enzyme components, including the HSPC117 ligase and La protein, in proficient initiation complexes, thus supporting coupling of several enzymatic activities for tRNA biosynthesis (Ramanathan A., Mani D., and Abu Ghannam N., unpublished data).…”

Regulation of Eukaryotic RNAPs Activities by Phosphorylation

“…Moreover, they all share five subunits (Rpb5, Rpb6, Rpb8, Rpb10, and Rpb12) with common functions but also with specific roles in their corresponding RNAPs (Cramer et al, 2008;Cuevas-Bermúdez et al, 2017). The structures of the three eukaryotic RNAPs, first solved in Saccharomyces cerevisiae, are highly conserved and their resolution has tremendously helped to understand the mechanism of transcription (Cramer et al, 2000;Armache et al, 2005;Engel et al, 2013;Fernandez-Tornero et al, 2013;Hoffmann et al, 2015;Sainsbury et al, 2015;Ramsay et al, 2020;Schier and Taatjes, 2020). The correct regulation of gene transcription depends on mechanisms that regulate the formation of large multiprotein complexes (RNAPs and their cognate factors) and their dynamics through all the transcription process.…”