PROFIT identifies proteins known to involve in mRNA biogenesis and environmentally responsive factors with known cytoplasmic functions.

(A) Schematic of the PROFIT approach. Following UV crosslinking and cell lysis, chromatin was fragmented by RNase-free DNase treatment. The Pol II–DNA–RNP ternary complex was immobilized via the FLAG-tagged Rpb3 subunit using anti-FLAG antibodies. Nascent RNA–binding proteins were released by on-column RNase I digestion and identified by mass spectrometry (MS). (B) Volcano plot of significant PROFIT hits. Proteins released by RNase I digestion of immobilized FLAG-tagged Pol II complexes were compared with a no-tag control strain. MS data from four biological replicates were analyzed using MaxQuant and Perseus. Statistical significance was assessed by a two-sample t-test with permutation-based FDR correction (FDR = 0.5, S0 = 0.1). Selected RNA-processing factors are indicated. (C) Distribution of significant hits classified as known RNA-processing factors (Battaglia et al., 2017) or other proteins. (D) Distribution of significant hits according to their reported ability to bind mature poly(A)+ mRNAs, based on published datasets (Beckmann et al., 2015; Mitchell et al., 2013 and this study. (E) Reproducibility of RNase I–released protein intensities across biological replicates. Pearson correlation coefficients are shown. (F) Hierarchical clustering and heat map of PROFIT LFQ intensities. Experimental and control samples segregate into distinct clusters. LFQ data were analyzed using Perseus following MaxQuant processing. Proteins detected in fewer than three replicates in at least one group were excluded; missing values were imputed from a normal distribution.

BioPROFIT combines PROFIT with proximity biotinylation to record transcription-proximal interactions and enables to follow the fate of the imprinting protein.

(A) Schematic of the BioPROFIT approach. As in PROFIT (Fig. 1), Pol II complexes were purified via Rpb3-FLAG. In addition, BirA (codon-optimized for S. cerevisiae) was fused to the C-terminal domain (CTD) of the Pol II subunit Rpb1. BioPROFIT signal therefore depends on both in vivo proximity-dependent biotinylation and in vitro Pol II purification. (B) Biotin as a molecular “history mark”. A PROFIT hit fused to eight AviTags is biotinylated when it resides in proximity to Rpb1-BirA during transcription. This design enables tracking of proteins that engage nascent transcripts and subsequently participate in post-transcriptional processes.

Rpg1 co-sediments with polysomes.

RPB9-BirA, RPG1-AviTag cells were grown in minimal biotin medium and labeled with biotin for 4 or 25 min, or continuously grown in YPD medium containing excess biotin. Cell extracts were subjected to sucrose-gradient polysome fractionation as described in Materials and Methods. (A) Polysome fractionation. Upper panels: polysome profiles. Lower panels: gradient fractions were subjected to western blot assay. Membranes were probed with IR-streptavidin (Bio-Rpg1-Avi), α-AviTag (bulk Rpg1-Avi), and α-Rpl1 antibodies (ribosomal protein L1). The position of the molecular weight marker is indicated. (B) Gradient profiles of Bio-Rpg1-Avi (right panel) and bulk Rpg1-Avi (left panel). Band intensities were quantified (Materials and Methods) and normalized such that each fraction represents a percentage of the total signal across the gradient. (C) Comparison of Bio-Rpg1-Avi and bulk Rpg1-Avi sedimentation. Profiles differ after 4 min of biotin labeling but converge at later time points. Ratios of normalized Bio-Rpg1-Avi to normalized bulk Rpg1-Avi were calculated for each fraction; a value of 1 indicates identical sedimentation.

Bio-Rpg1-AviTag co-sediments with polysomes in a puromycin-sensitive manner.

(A) Rpb9-BirA, Rpg1-AviTag cells were grown in minimal biotin medium and labeled with biotin for 10 min prior to harvest. Cell lysates were divided into untreated and puromycin-treated (1.6 mM, 15 min) samples and subjected to sucrose-gradient polysome fractionation. Upper panels: polysome profiles. Lower panels: gradient fractions were subjected to western blot assay. Membranes were probed with IR-streptavidin (Bio-Rpg1-Avi), α-AviTag (bulk Rpg1-Avi), α-Rpl1 (ribosomal protein L1), and α-Pab1 antibodies. The position of the molecular weight marker is indicated. (B–D) Quantification of gradient fractions. Band intensities were normalized to the total signal per gradient and are presented as percentages.

PROFIT and BioPROFIT reveal heat-shock-induced co-transcriptional binding of Ssa2 to nascent RNA.

(A) PROFIT analysis of heat-stressed versus non-stressed cells. Cells were grown to mid-log phase and either harvested directly or shifted to 40 °C for 30 min prior to harvest. Cell grindates were subjected to PROFIT analysis (see Materials and Methods and Fig. 1A–B). Arrows indicate Ssa1 (yellow) and Ssa2 (red). (B) BioPROFIT identification of biotinylated Ssa2 (Bio-Ssa2) associated with Pol II (right) or nascent RNA (middle). Cells were grown in minimal biotin medium at the optimal temperature and either harvested directly or subjected to heat shock (40 °C, 30 min). Biotin (1 µM) was added for 15 min before harvest. Cells were processed as in Fig. 1A. Following RNase treatment and analysis of the release proteins (“Nascent RNA associated”), the RNP-depleted Pol II complex, remained bound to the column, was eluted with FLAG peptide (“Pol II–associated”). Proteins were analyzed by western blot. The same membrane was decorated with IR-streptavidin to detect Bio-Ssa2-Avi; with anti-Hsp70 antibodies to detect Ssa2-Avi and other Hsp70s (distinguished by their electrophoretic mobility); anti-FLAG antibodies to detect Rpb3-FLAG; and with anti-Rpb4 antibodies to detect Rpb4. (C) Quantification of Bio-Ssa2 associated with nascent RNA and Pol II. Bio-Ssa2 was detected using IR-streptavidin, and Rpb3-FLAG was detected using anti-FLAG antibodies. Bio-Ssa2 values were normalized to the corresponding Rpb3-FLAG signal at each temperature. For each sample pair, the maximal value was set to 100%. (D) Quantification of Rpb4 associated with nascent RNA and Pol II, normalized to Rpb3-FLAG as in (C).

Rpb4 is required for co-transcriptional binding of multiple PROFIT hits.

(A) Volcano plot comparing PROFIT results from WT and rpb4Δ cells. Grindates of RPB3-FLAG cells carrying or lacking RPB4 were subjected to PROFIT analysis as described in Fig. 1A and in Materials and Methods. Statistical analysis of two biological replicates (each including experimental and no-tag controls) was performed using Perseus. (B) BioPROFIT analysis showing that efficient co-transcriptional binding of Spt6-Avi to nascent RNA depends on Rpb4. Cells, genotypes of which are indicated (“mutant” refers to or rpb4E19D, E20D, E21D, E22D), were grown in minimal biotin medium under optimal conditions. Biotin (1 µM) was added for 15 min before harvest. Cells were processed as in Fig. 1A. Following RNase treatment and analysis of the release proteins (“Nascent RNA associated”), the RNP-depleted Pol II complex, remained bound to the column, was eluted with FLAG peptide (“Pol II–associated”). Proteins were analyzed by western blot. The same membrane was decorated with IR-streptavidin to detect Bio-Spt6-Avi; anti-FLAG antibodies to detect Rpb3-FLAG; and with anti-Rpb4 antibodies to detect Rpb4. The bulk Spt6 was detected by anti-HIS Abs that bound to (HIS)x6, which was placed upstream of the FLAG.

BirA fused to Pol II subunits specifically biotinylates AviTagged proteins.

(A-B) Fusion of BirA to Rpb1 shifts electrophoretic mobility (A) without compromising cell growth (B). BirA was surgically inserted at the endogenous RPB1 (A, B, D, E) or RPB9 (C) loci, leaving both 5’ and 3’ non-coding regions unpurterbed. (C) Rpg1-AviTag and Tif4631-AviTag are biotinylated by Rpb9-BirA following biotin addition. Cells were lysed under harsh denaturing conditions (including 50% urea, 10% SDS) to prevent post-lysis enzymatic activity and analyzed by western blotting. (D) Five minutes of biotin labeling is sufficient to detect AviTagged Ssa1 or Ssa2 in strains expressing Rpb1-BirA, but not NLS-GFP-BirA. Bulk Ssa1 was detected with anti-AviTag antibody, while biotinylated Ssa1 (bioSsa1) was detected using IR-Streptavidin (see Materials and methods). (E) Xrn1-AviTag is biotinylated in vivo.

Translation inhibition does not alter Rpg1 biotinylation.

(A) Rpb9-BirA Rpg1-AviTag cells were treated with cycloheximide (CHX) either before or after biotin labeling. Cells were lysed under denaturing conditions (50% Urea 10% SDS) to block enzymatic activity and analyzed by immunoblotting. (B) Band intensities were quantified (Materials and Methods), normalized to total signal, and used to calculate the ratio of biotinylated to total Rpg1. Data represent mean ± SD from two biological replicates.