Structure of human RNA Polymerase III

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>In eukaryotes, RNA Polymerase (Pol) III is the enzyme specialised for the transcription of the entire pool of tRNAs and several other short, essential, untranslated RNAs. Pol III is a critical determinant of cellular growth and lifespan across the eukaryotic kingdom. Upregulation of Pol III transcription is often observed in cancer cells and causative Pol III mutations have been described in patients affected by severe neurodevelopmental disorders and hypersensitivity to viral infection.</jats:p><jats:p>Harnessing CRISPR-Cas9 genome editing in HeLa cells, we isolated endogenous human Pol III and obtained a cryo-EM reconstruction at 4.0 Å. The structure of human Pol III allowed us to map the reported genetic mutations and rationalise them. Mutations causing neurodevelopmental defects cluster in hotspots that affect the stability and/or biogenesis of Pol III, thereby resulting in loss-of-function of the enzyme. Mutations affecting viral sensing are located in the periphery of the enzyme in proximity to DNA binding regions, suggesting an impairment of Pol III cytosolic viral DNA-sensing activity.</jats:p><jats:p>Furthermore, integrating x-ray crystallography and SAXS data, we describe the structure of the RPC5 C-terminal extension, which is absent in lower eukaryotes and not visible in our EM map. Surprisingly, experiments in living cells highlight a role for the RPC5 C-terminal extension in the correct assembly and stability of the human Pol III enzyme, thus suggesting an added layer of regulation during the biogenesis of Pol III in higher eukaryotes.</jats:p>

Specific Features of RNA Polymerases I and III: Structure and Assembly

“…Recently published structures of human RNAPIII revealed a high level of conservation (Ramsay et al, 2020;Li et al, 2021;Wang et al, 2021). Moreover, mutations of specialized RNAPs lead to genetic disorders, such as Treacher-Collins syndrome and hypomyelinating leukodystrophy (Ramsay et al, 2020;Girbig et al, 2021), demonstrating the requirement for precise coordination among all three RNAPs and their assembly. Research on RNAPIII assembly in yeast focused on rpc128-1007 mutations that disturbed the interface between the Rpc128 and Rpc40 subunits.…”

“…Research on RNAPIII assembly in yeast focused on rpc128-1007 mutations that disturbed the interface between the Rpc128 and Rpc40 subunits. Interestingly, multiple disease-associated mutations of human RNAPIII subunits tend to cluster within the region of the RNAPIII assembly platform, suggesting that defects in RNAPIII biogenesis may have severe health consequences (Ramsay et al, 2020;Girbig et al, 2021). Further studies of RNAP assembly should reveal additional factors that are involved in this process and improve our understanding of this vital pathway.…”

The overexpression of FKBP4 in patients with lung adenocarcinoma predicts poor prognosis and tumor progression

“…Basic transcription factors are necessary for the initiation of RNA polymerase II transcription and can maintain the basic level of transcription. Once the basic level of transcription is imbalanced, it will affect the function of RNAs, and then induce the occurrence and progression of tumors [19,20]. Proteasome is a multi-protein organelle that participates in cellular proteostasis through destroying damaged or short-lived proteins.…”

The human RNA polymerase I structure reveals an HMG-like transcription factor docking domain specific to metazoans

“…In higher eukaryotes, regulatory variations dependent on tissue type, developmental state and cell-cycle stage are adding additional layers of complexity. The structure-function analysis of human Pol II 3 and Pol III [4][5][6][7] showed both similarities in the catalytic mechanisms and divergence in regulatory elements among organisms.…”