A constant level of Pol III is maintained in the nucleus by nuclear retention and degradation
Description
Pol III functions in the nucleus for transcription and cytoplasm as a viral-DNA sensor, but how cells maintain the cytoplasmic-to-nuclear partitioning of Pol III activity is unknown. Here, we found that Pol III forms transcription-dependent protein clusters in the nucleus and preferentially associates with assembly factors in the cytoplasm. Acute protein degradation of Pol III subunits followed by transient protein labeling showed that nuclear and cytoplasmic Pol III response differently after the loss of their subunits. In this file, two parts are involved. The first part is for image of western blot and another part is for imaging. The HSP_inhibit result refers to Celastrol (inhibitor of HSP90) or VER-155008 (inhibitor of HSP70), the cell was treated with HSP70/HSP90 inhibitor respectively and fractionated to the cytoplasm and the nucleus (CN), six RNA polymerase III subunits were blotted and Lamin B1 as an indicator of nuclear fractions and α-Tubulin as an indicator of cytoplasmic fractions. RPC2_Degradation_TimeCourse result refers to auxin induced degradation of RPC2. In this folder, we generated the RPC2-degron cell line, and treated cell with auxin to induce RPC2 degradation. LMB_MG132 result refers to Leptomycin B(LMB) and MG132 treatment of RPC1-degron. IP_WB result refers to immunoprecipitate of RPC1, RPC2, RPC3. In this folder, we use RPB5-degron and RPC2-degron cells. The cells were treated with auxin to induce RPB5 or RPC2 degradation, and the cytoplasm or nuclear fractions were immunoprecipitated for western blot. The another part is Figure_Imaging, which include 5 folders. 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). Figure2C_6h_degradation result refers Live-cell imaging. 4 cell lines include RPC1-degron, RPC2-degron, RPAC1-degron and RPB5-degron were used here. The cells were with auxin induction for 6 h, and RPC1-Halotag, RPC2-Halotag ,RPC3-Halotag were imaged. Figure3D_HSP_inhibitor result refers Live-cell imaging of RPC1-degron; RPC2-HaloTag (C1DC2H). the C1DC2H cell was treat with auxin, Celastrol (inhibitor of HSP90) or VER-155008 (inhibitor of HSP70), and RPC1-GFP and RPC2-Halotag was imaged. Figure3A+3C_LMB_MG132_treatment result refers Live-cell imaging of (C1DC2H. The C1DC2H cell was treat with auxin, LMB,MG132 and RPC1-GFP and RPC2-Halotag was imaged. FigureS1C_TF_inhibitor result refers Live-cell imaging of C1DC2H. The C1DC2H cell was treat with auxin, a-amanitin(Pol II inhibitor), ML60218(Pol III inhibitoor), Actinomycin D (pan-transcription inhibitor), and RPC1-GFP and RPC2-Halotag was imaged. Figure4_Folder refers Live-cell imaging of C1DC2H. The C1DC2H cell was treat with auxin for RPC1-degradation, and we removed the auxin for RPC1 rescue. During that, RPC2-HaloTag was treated with JF646 ligand to label the preexisting RPC2 (old) and with JF549 ligand to label the newly synthesized RPC2 (new).