Pol II Preferentially Regulates Ribosomal Protein Expression by Trapping Disassociated Subunits

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.