Fig. 2: RPB7 interaction with RPB1 and its dimerization are associated with the stability of nuclear RPB1.
From: CTDP1 and RPB7 stabilize Pol II and permit reinitiation
a Schematic of the design of RPB7 mutants, based on the protein secondary structure provided by Uniport. Right: Western blot analyses of anti-FLAG immunoprecipitates collected from lysate of exogenous RPB7-FLAG-expressing 293T cells. FLAG served as the IP fraction loading control. b Visualization of the RPB7 structure (blue) in the Pol II complex (PDB: 6xre) and its structural alignment with DEL-RPB7 (pink), predicted by AlphaFold293. c Schematic of exogenous expression of wild-type (WT) or mutant RPB7 at the Tigre locus in RPB7 degron cells is shown. The loop region was deleted in DEL-RPB7, and the NLS tag “MAPKKKRKVGIHGVPAA” was fused at its N-terminus to generate the NLS-DEL-RPB7 construct. The dimerization mutant (mDimer) was designed according to the sequence alignment of mouse RPB7 and yeast RPB7 mutant previously reported51. d Western blotting analyses for these four Tigre knock-in cells in the presence and absence of auxin treatment. β-Actin served as the loading control. Right: Fold changes in RPB1 protein levels (n = 3, biological replicates) are shown as means ± SD. Statistical significance, two-sided unpaired t-test; **p < 0.01. L-R p value: 0.0021, 0.0015, and 0.0012. e Representative fluorescence signals of these four Tigre knock-in RPB7 (green) degron cells, which were stained with Janelia Fluor 549 HaloTag ligand (red) to visualize the exogenously expressed RPB7 and Hoechst to indicate nuclei (DNA, blue). Scale bar, 20 μm. f Nuclear signals of exogenous RPB7 in (e) (n = 15, biological replicates) are shown as means ± SD. Statistical analyses were performed by compared each RPB7 mutant with wild-type RPB7 under Auxin 0 h condition, two-sided unpaired t-test; ****p < 0.0001; *p < 0.05. L-R p value: 9.242 × 10−11, 0.0402 and 1.145 × 10−10. g The loop regions from functionally equivalent subunits of RPB7 (RPC8 and RPA43) or yeast are shown. The right panel shows the western blot analyses in RPB7 mutants-expressing RPB7 degron cells. h Fold changes in RPB1 protein levels in (g) (n = 2, biological replicates) are shown as means.
