Fig. 1: Localization of TBPL1 and PAF1 with nucleolar Pol II at the rDNA IGS.

a Schematic of compBioID used to identify nucleolar Pol II interactome. Expression of miniTurbo- RPB9 promotes biotinylation of proximal proteins. This is followed by sucrose gradient-based (SG) enrichment of nucleoli and identification of biotinylated factors using mass spectrometry. Non-nucleolar enriched whole-cell samples served as control. b Heatmap showing peptide counts for biotinylated proteins detected in nucleolar-enriched (NOL), whole-cell (WC), or both samples. c Top 19 biotinylated proteins detected only in NOL samples and categorized under nucleic acid regulation using STRING analysis. List of internal controls detected known to be nucleolar, non-nucleolar, or both. Peptide counts are shown with the Bayesian False discovery rate (BFDR). d Biological processes gene ontology analysis of the 19 proteins from panel (c) with the number of proteins in the molecular network (bubble size), and the strength of the enrichment (color). e Immunoblots showing Pol II co-immunoprecipitates with PAF1 or TBPL1. f Schematic of modified SG-based cellular fractionations to compare nucleoplasmic+cytoplasmic (Nuc+Cyto), nucleoplasmic (Nuc), Cytoplasmic (Cyto), and NOL fractionations to non-fractionated WC samples by Western blotting (WB). g, h Immunoblots assessing the presence of PAF1, FBL, TBPL1, DR1, and MED26 in WC, Nuc+Cyto, Nuc, Cyto, and NOL samples. Nucleolar-enriched FBL served as positive control and non-nucleolar proteins β-actin, vinculin, and LDHA served as negative controls. i Schematic of a mammalian rDNA unit containing the 47S pre-rRNA-encoding 13.3 kb rRNA gene and the 29.7 kb intergenic spacer (IGS, orange) with the positioning of key primer pairs used in this study. Also shown are IGS sites harboring TCT motifs. j Chromatin immunoprecipitation (ChIP) showing the localization of TBPL1 (left) and PAF1 (right) at the IGS. 28S rRNA-coding site served as negative control. k ChIP-re-ChIP showing the co-localization of TBPL1 (left) or PAF1 (right) with Pol II at the IGS. Enrichments are presented relative to the Pol II+immunoglobulinG (IgG) mock ChIP-re-ChIP control. 28S rRNA-coding site served as negative control. a–k Experiments were performed using Flp-In 293 T-REx^TM (b–d) or HEK293T cells (e–k); blots (e, g, h) are representative of three biologically independent experiments; data in (j, k) are shown as mean  ± s.d., were from large experimental sets sharing IgG control, were analyzed using two-way ANOVA, and n = 3 (j) or n = 4 (k) biologically independent experiments. Source data are provided as a Source Data file.