Fig. 5: CTD with linker region of RPB1 is required for its destabilization.

a Schematic of exogenous expression of wild-type (WT) and mutated RPB1 are shown. The amino acid from position 1593 to the last amino acid on RPB1 represents “CTD.” The amino acid from position 1486 to position 1592 on RPB1 represents the “linker region.” Mutant “m26A” represents the RPB1 mutant with all serine and threonine in the linker region mutated to alanine, while “m7A” represents the RPB1 mutant with seven amino acids in the linker region mutated to alanine, which have been reported to have phosphorylation modifications⁷¹. b Western blot analyses of anti-FLAG immunoprecipitates collected from lysate of exogenous RPB1-expressing cells, which revealed their comparable interaction with other Pol II subunits, like RPB7 and RPB3 shown here. Western blot analyses of mutant RPB1 levels in whole-cell lysates after RPB7 (c) or CTDP1 (e) depletion. GFP served as the degradation control, FLAG represented the protein levels of exogenous RPB1 and β-Actin served as a loading control. The fold changes of exogenous RPB1 fluorescence intensity (median, n = 3, biological replicates) upon depletion of RPB7 (d) or CTDP1 (f) are shown. Exogenous RPB1-expressing cells were labeled by Janelia Fluor 549 HaloTag ligand, then detected by flow cytometer. Statistical significance, two-sided unpaired t-test; ****p < 0.0001; error bars, SD. d L-R p value: 1.078 × 10⁻⁸, 9.895 × 10⁻⁹, 1.932 × 10⁻⁶, 1.202 × 10⁻⁶, 1.682 × 10⁻⁷, and 2.109 × 10⁻⁸. f L-R p value: 7.428 × 10⁻⁹, 2.010 × 10⁻⁸, 2.252 × 10⁻⁵, 3.973 × 10⁻⁵, 2.679 × 10⁻⁷, and 0.0006. g, h Representative fluorescence signals of exogenous RPB1-expressing cells treated with or without auxin for 3 h. GFP channel (green) represented RPB7 (g) or CTDP1 (h) and served as the degradation control. Cells were stained with Janelia Fluor 549 to visualize the exogenously expressed RPB1 (red) and Hoechst was used to indicate nuclei (DNA, blue). Scale bar, 20 μm.