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.
Highlights
In eukaryotes, RNA Polymerase (Pol) III is specialized for the transcription of tRNAs and other short, untranslated RNAs
Fractionation experiments followed by immunopurification using an anti-green fluorescent protein (GFP) nanobody revealed that Pol III is present in both nuclear and cytoplasmic fractions (Fig. 1b), in agreement with previous reports highlighting a Pol III cytosolic DNA-sensing activity[17,18]
The structure confirms the overall high-degree of structural homology with its S. cerevisiae counterpart and highlights specific differences, such as a rearranged foot domain (Supplementary Fig. 5)
Summary
RNA Polymerase (Pol) III is specialized for the transcription of tRNAs and other short, untranslated RNAs. To obtain a high-resolution structure of human Pol III, we isolated the endogenous complex from HeLa cells. The majority of imaged particles represented the intact 17-subunit human Pol III but a sizeable fraction with a similar angular distribution displayed no density for the RPC3/6/7 heterotrimer, which had possibly dissociated during purification, in agreement with earlier reports[19], or during cryo-EM specimen preparation.
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