Pol II Preferentially Regulates Ribosomal Protein Expression by Trapping Disassociated Subunits
Description
Pol II has been recognized as a passively regulated holoenzyme. Here, fractions containing disassociated RPB3 (dRPB3) were identified by size exclusion chromatography in various cells. Through a unique strategy, i.e., Specific Degradation of Disassociated Subunits (SDDS), we showed that dRPB3 functions as a regulatory component of Pol II to enable the preferential control of 3’ end processing of ribosomal protein genes. Figure1A show Western blot validation of isolated chromatin fractions from native or cross-linked mESCs. Figure1B show Western blot analysis of indicated aliquots of fractions from the native or cross-linked mESC chromatin extracts isolated by size exclusion chromatography. Figure1C show Western blot analysis of different cross-linked human cell lines, similar to Figure1B. Figure2C show Western blot analysis of indicated proteins in RPB1 IP samples from wild-type, RPB1-CTD and dRPB3 degron mESCs. Figure2D show Western blot analysis of different phosphorylation forms of RPB1 in wild-type and dRPB3 degron mESCs. Figure2E show the Immunofluorescence analysis of the distribution of the RPB1 signal (red), and DAPI staining indicates the nucleus. Figure2F show the Western blot analysis in fractionated chromatin extracts after size exclusion chromatography with or without dRPB3 degradation. Figure2G show the Western blot analysis of interaction between RPB11 and RPB1 after RPB3 or dRPB3 degradation. Figure2H show the comparable structures of dRPB3 and RPB3 in RPB1-RPB3 fusion protein predicted by AlphaFold2. Figure4C show the Western blot results of two immunoprecipitation assay. One examines the NCBP1, PCIF1 in IP samples with GFP antibody from indicated mESCs , the other examines NCBP1, PCIF1, PCF11 in IP samples with RPB1 anditody from RPB3 degron mESCs with or without IAA treatment. Figure4D show the Western blot analysis of NCBP1, PCIF1, PCF11 in IP samples with RPB1 antibody from dRPB3 degron mESCs with or without IAA treatment. Figure5A and Figure5B show the Western blot analysis of NCBP1, PCIF1 in IP samples with RPB3 antibody from RPB1-NTD degron and RPB1-CTD degron mESCs with or without IAA treatment. Figure5C and Figure6B show the Western blot analysis of the interaction between NCBP1 or ELL2 with different RPB3 truncation forms. Figure5D and Figure6C show the Western blot validation of the direct interaction between NCBP1 or ELL2 with full length or truncated RPB3 using GST-pulldown assay. Figure5F and Figure6D show the Western blot validation of protein expression in different treatment groups. FigureS1C show the images of PCR-based genotyping of each degron mESCs, with wild-type mESCs as control. FigureS1D show the Western blot analysis of interaction between indicated subunits and RPB1 after RPB3, dRPB3 or RPB5 degradation. FigureS3G show the Western blot validation in dRPB10 and dRPB11 degron mESCs. FigureS4E show the co-localization analysis of purified RPB3 and CTD droplets in vitro, with mCherry as the control.
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Steps to reproduce
For the chromatin fraction isolation followed by size exclusion chromatography, the native or cross-linked cells were lysed in NP-40 lysis buffer, and centrifuged. The nuclei pellets were lysed in lysis buffer, and centrifuged. The supernatant contains the nucleoplasmic fraction, and the pellets were digested by Benzonase, DNase I, and RNaseA for 4 hour RT. Then the free chromatin fractions were fractionated by Superdex 200 Increase column. For the ChIP-Western assay, the chromatin extracts were isolated in cross-linked mESCs according to the ChIP-seq assay, and indicated antibodies were used to enrich the target proteins, and Western blot assay were conducted to examine the protein interaction change. For the Co-IP assay, the HEK293T cells were co-transfected with expression constructs. And 2 days later, the cell lysate were collected and incubated with GFP-Trap beads to enrich the target protein, and analyze the protein interaction strength by Western blot assay. For the GST-pulldown assay, the NCBP1, ELL2, GST-RPB3, and GST-RPB3-truncation form were purified by using specific agarose based on the covalent tags, and further purified by size exclusion chromatography using Superdex 75 FPLC gel filtration column. To perform GST-pulldown assay, glutathione agarose beads bound with GST-tagged proteins were incubated with NCBP1 or ELL2 in 4℃ with rotations. After washing, the beads were denatured in protein loading buffer at 100℃ , and the interaction was detected by Western blot.