Subcellular Localization Shapes the Fate of RNA Polymerase III
Description
RNA Pol III plays a vital role in transcription and as a viral-DNA sensor, but how it is assembled and distributed within cells remain poorly understood. Here, we show that Pol III is assembled with chaperones in the cytoplasm and forms transcription-dependent protein clusters upon transport into the nucleus. The largest subunit (RPC1) depletion through an auxin-inducible degron leads to rapid degradation and disassembly of Pol III complex in the nucleus and cytoplasm, respectively. This generates a pool of partially assembled Pol III intermediates, which can be rapidly mobilized into the nucleus upon the restoration of RPC1. Our study highlights the critical role of subcellular localization in determining Pol III's fate and provide insight into the dynamic regulation of nuclear Pol III levels and the origin of cytoplasmic Pol III complexes involved in mediating viral immunity. In this file, the result of Western blot and Cell Imaging was listed as Figures shown. A brief message of this folder was characterized as below: The mES cells were treated with Doxycycline, auxin, Leptomycin B (LMB), MG132, Celastrol (an HSP90 inhibitor), VER-155008 (an HSP70 inhibitor), α-amanitin (a Pol II inhibitor), ML60218 (a Pol III inhibitor), and Actinomycin D (a pan-transcription inhibitor) at the common used concentration and treatment time. The details can be found in the methods section of our paper. The Raw image was captured using Andor's Dragonfly microscope with .ims type, and it could be opened by imaris (oxford instrument) or ImageJ (FiJi). In the folder, some abbreviation were indicated as： TC: time course; CN: cytoplasm and nuclear fraction; LMinh: LMB and/or MG132 inhibitor treatment; BIOT: halotag biotin ligand