Extended Data Fig. 5: CTD length affects RNAPII subcellular distribution and expression.

a, Diffusion coefficient histogram for RNAPII with varying CTD lengths (n: number of trajectories; mean value ± s.d.). b-c, b, nuclear/cytoplasmic ratio and c, relative nuclear intensity of WT RNAPII and CTD mutants (median (line), IQR (box), 10–90 percentile (whiskers) and outliers (crosses), n = 95 nuclei for CTD0, 52 nuclei for CTD8, 69 nuclei for CTD9, 64 nuclei for CTD10, 112 nuclei for CTD15, 108 nuclei for CTD20, 69 nuclei for CTD26, 73 nuclei for CTD52, 91 nuclei for CTD78, 102 nuclei for CTD104). CTD truncation led to increased cytoplasmic and nucleolar distribution, and overexpression of RNAPII. d, Amount of bound RNAPII normalized to nuclear fluorescence intensity (n = 100 resamplings; mean value ± s.d.). The normalized value is an approximation, as the relationship between nuclear fluorescence intensity and RNAPII levels may not be strictly linear. e, Fluorescence signal of RPB1^Rpo21-AID*-miRFP670nano3 (red) before and after RNAPII degradation by auxin. Nucleolar and ER markers in green. Scale bar: 1.0 μm. f, Western blot showing time course degradation of RPB1^Rpo21-AID* by auxin, detected using CTD antibody (top), and Ponceau S staining on total protein (bottom). g,h, Cells expressing ectopic RPB1^Rpo21-Halo in an RPB1^Rpo21 AID background show minimal changes in the g, bound fraction (n = 100 resamplings; mean value ± s.d.; two-tailed unpaired t-test, * P = 0.015275) and h, f_180/0 of RNAPII after auxin treatment, compared to those in WT expressing RPB1^Rpo21-Halo (n = 100 resamplings; mean value ± s.d.). i, Spot assay for strains with prd5Δ, GFP markers (GGEGp), RPB1^Rpo21-nCDT26, or RPB1^Rpo21-nCDT26-Halo showing identical growth. j, Spot assay demonstrating impaired cell growth for RPB1^Rpo21 or MED14^Rgr1 AID degron. All AID* constructs contain miRFP670nano3 as a fluorescence marker. k, Spot assay for strains with HaloTag fusions to various PIC component subunits to assess MPA sensitivity. Ssl2-Halo (TFIIH), Halo-Ssl2 (TFIIH), Tfa2-Halo (TFIIE), Tfg2-Halo (TFIIF) exhibited MPA sensitivity. Source numerical data and unprocessed blots are available in source data.

Source data