Fig. 1: A systematic assay expands the network of co-translational interactions in TFIID and identifies nascent TAF1 polypeptide as a central hub in the assembly process.
From: Hierarchical TAF1-dependent co-translational assembly of the basal transcription factor TFIID
a, Schematic structure of TFIID. Half-circle subunits represent HFD partners. b, RNA immunoprecipitation (RIP) assays using an antibody against endogenous human TAF10 on HeLa cell polysome extracts. Potential target mRNAs were tested by RT–qPCR. Data points correspond to technical duplicates from n = 3 biological replicates. c, RIP-coupled RT–qPCR assays using an antibody against endogenous mouse TAF10, performed on mESCs. Data points represent technical duplicates from n = 2 biological replicates. d, Schematic representation of the GFP-RIP-coupled RT–qPCR assay using HeLa cell lines expressing doxycycline (Dox)-inducible GFP-TAFs to systematically probe co-translational assembly in TFIID. e, Matrix summarizing the results of the systematic GFP-RIP assay in d. GFP-tagged TFIID subunits were used as baits in a GFP-RIP assay from polysome extracts (rows), and enrichment for TFIID-subunit mRNAs was assessed by RT–qPCR (columns). The area of each circle is proportional to mRNA log2(fold enrichment (FE)) over mock IP. Combinations whose FE was less than fourfold of that of negative control target mRNA (PPIB) are not shown in the plot and are considered negative. Gray circles represent hits for bait mRNA. Black circles represent Co-TA hits. Red circles highlight the widespread enrichment for TAF1 mRNA from RIP of several TFIID subunits. Stars indicate subunits for which GFP fusion resulted in ambiguous protein functionality. Results represent the mean of n = 2 biological replicates. f, RIP-coupled RT–qPCR assays against endogenous TAF6 performed on HeLa cells. The C-terminal location of the epitope prevented the detection of the nascent TAF6 protein, along with its own mRNA, and the simultaneous co-TA with TAF9 (TAF6 HFD partner). Data points correspond to technical triplicates from n = 2 biological replicates. g, RIP-coupled RT–qPCR assays against endogenous TAF7. Data points correspond to technical triplicates from n = 2 biological replicates. Bar graphs in the figure show the mean of the data. Antigen regions for the antibodies used are indicated. HFD, histone-fold domain; HEAT, HEAT-repeat domain; TAF1iD, TAF1-interaction domain; FRT-TO, FLP Recombination Target-Tet-ON; Ptet, tetracycline-responsive promoter. Cycloheximide (CHX) prevents ribosome dissociation from the mRNA. By contrast, puromycin (Puro) induces premature nascent polypeptide chain termination and release from the ribosome or mRNA.
