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The Protein Interaction Network of the Human Transcription Machinery Reveals a Role for the Conserved GTPase RPAP4/GPN1 and Microtubule Assembly in Nuclear Import and Biogenesis of RNA Polymerase II

2010; Elsevier BV; Volume: 9; Issue: 12 Linguagem: Inglês

10.1074/mcp.m110.003616

ISSN

1535-9484

Autores

Diane Forget, Andrée-Anne Lacombe, Philippe Cloutier, Racha Al‐Khoury, Annie Bouchard, Mathieu Lavallée‐Adam, Denis Faubert, Celia Jerónimo, Mathieu Blanchette, Benoit Coulombe,

Tópico(s)

Nuclear Structure and Function

Resumo

RNA polymerase II (RNAPII), the 12-subunit enzyme that synthesizes all mRNAs and several non-coding RNAs in eukaryotes, plays a central role in cell function. Although multiple proteins are known to regulate the activity of RNAPII during transcription, little is known about the machinery that controls the fate of the enzyme before or after transcription. We used systematic protein affinity purification coupled to mass spectrometry (AP-MS) to characterize the high resolution network of protein interactions of RNAPII in the soluble fraction of human cell extracts. Our analysis revealed that many components of this network participate in RNAPII biogenesis. We show here that RNAPII-associated protein 4 (RPAP4/GPN1) shuttles between the nucleus and the cytoplasm and regulates nuclear import of POLR2A/RPB1 and POLR2B/RPB2, the two largest subunits of RNAPII. RPAP4/GPN1 is a member of a newly discovered GTPase family that contains a unique and highly conserved GPN loop motif that we show is essential, in conjunction with its GTP-binding motifs, for nuclear localization of POLR2A/RPB1 in a process that also requires microtubule assembly. A model for RNAPII biogenesis is presented. RNA polymerase II (RNAPII), the 12-subunit enzyme that synthesizes all mRNAs and several non-coding RNAs in eukaryotes, plays a central role in cell function. Although multiple proteins are known to regulate the activity of RNAPII during transcription, little is known about the machinery that controls the fate of the enzyme before or after transcription. We used systematic protein affinity purification coupled to mass spectrometry (AP-MS) to characterize the high resolution network of protein interactions of RNAPII in the soluble fraction of human cell extracts. Our analysis revealed that many components of this network participate in RNAPII biogenesis. We show here that RNAPII-associated protein 4 (RPAP4/GPN1) shuttles between the nucleus and the cytoplasm and regulates nuclear import of POLR2A/RPB1 and POLR2B/RPB2, the two largest subunits of RNAPII. RPAP4/GPN1 is a member of a newly discovered GTPase family that contains a unique and highly conserved GPN loop motif that we show is essential, in conjunction with its GTP-binding motifs, for nuclear localization of POLR2A/RPB1 in a process that also requires microtubule assembly. A model for RNAPII biogenesis is presented. Significant effort has been made over the past 4 decades to identify and characterize the factors that regulate the activity of RNA polymerase II (RNAPII), 1The abbreviations used are:RNAPRNA polymeraseAPaffinity purificationCCTchaperonin containing TCP-1IRinteraction reliabilityLMBleptomycin BNESnuclear export signalNLSnuclear localization signalRPAPRNAPII-associated proteinTAPtandem affinity purificationYcyeast complete medium5FOA5-fluoroorotic acid. the eukaryotic enzyme that synthesizes mRNA and several non-coding RNA. A myriad of protein factors have the ability to regulate the activity of RNAPII during the act of transcription. DNA-binding transcriptional regulators are known to control the activity of the RNAPII transcription machinery in a gene- and cell type-specific manner (1.Ptashne M. Gann A. Transcriptional activation by recruitment.Nature. 1997; 386: 569-577Crossref PubMed Scopus (934) Google Scholar, 2.Carey M. 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RNA polymerase affinity purification chaperonin containing TCP-1 interaction reliability leptomycin B nuclear export signal nuclear localization signal RNAPII-associated protein tandem affinity purification yeast complete medium 5-fluoroorotic acid. Quite surprisingly and despite extensive efforts to analyze the regulatory mechanisms targeting transcription and transcription factors themselves, very little is known about the molecular machinery that regulates the fate of RNAPII before and after transcription. For example, the process of biogenesis of the three nuclear RNAPs (RNAPI, -II, and -III), which comprise both common and specific subunits, has been the subject of only a few reports (16.Hardeland U. Hurt E. Coordinated nuclear import of RNA polymerase III subunits.Traffic. 2006; 7: 465-473Crossref PubMed Scopus (17) Google Scholar). We hypothesized that the protein complexes involved in the assembly, folding, and nuclear import of RNAPII are likely to be found in the human cell soluble fraction as opposed to the insoluble fraction that contains chromatin and actively transcribing RNAP molecules. We therefore conducted a survey of the soluble protein complexes that associate with RNAPII using protein affinity purification coupled to mass spectrometry (AP-MS) to identify the factors involved in the biogenesis of RNAPII. Twenty-eight tagged proteins were purified, and their associating partners were identified by MS. High confidence interactions were selected computationally and then used to draw a map of the interactions connecting these complexes. The composition and organization of this network revealed important features about the eukaryotic transcriptional machinery. Most notably, the highly conserved GTPase RNAPII-associated protein 4 (RPAP4)/GPN1 was found to have multiple interactions with the subunits of RNAPs, tubulins, and components of the microtubule assembly machinery, including the chaperonins (chaperonin containing TCP-1 (CCT) complex) and prefoldins (prefoldin-like complex). Our results indicate that both RPAP4/GPN1 activity and microtubule assembly/integrity are required for nuclear localization of the largest RNAPII subunits, POLR2A/RPB1 and POLR2B/RPB2. Selected human polypeptides were cloned into the mammalian expression vector pMZI (17.Zeghouf M. Li J. Butland G. Borkowska A. Canadien V. Richards D. Beattie B. Emili A. Greenblatt J.F. Sequential Peptide Affinity (SPA) system for the identification of mammalian and bacterial protein complexes.J. Proteome Res. 2004; 3: 463-468Crossref PubMed Scopus (152) Google Scholar) carrying a TAP tag at its C terminus (18.Puig O. Caspary F. Rigaut G. Rutz B. Bouveret E. Bragado-Nilsson E. Wilm M. Séraphin B. The tandem affinity purification (TAP) method: a general procedure of protein complex purification.Methods. 2001; 24: 218-229Crossref PubMed Scopus (1420) Google Scholar, 19.Rigaut G. Shevchenko A. Rutz B. Wilm M. Mann M. Séraphin B. A generic protein purification method for protein complex characterization and proteome exploration.Nat. Biotechnol. 1999; 17: 1030-1032Crossref PubMed Scopus (2280) Google Scholar). Stable human embryonic kidney cell lines (EcR-293; derived from HEK293) carrying these constructs were produced as described previously (20.Jeronimo C. Forget D. Bouchard A. Li Q. Chua G. Poitras C. Thérien C. Bergeron D. Bourassa S. Greenblatt J. Chabot B. Poirier G.G. Hughes T.R. Blanchette M. Price D.H. Coulombe B. Systematic analysis of the protein interaction network for the human transcription machinery reveals the identity of the 7SK capping enzyme.Mol. Cell. 2007; 27: 262-274Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar, 21.Cloutier P. Al-Khoury R. Lavallée-Adam M. Faubert D. Jiang H. Poitras C. Bouchard A. Forget D. Blanchette M. Coulombe B. High-resolution mapping of the protein interaction network for the human transcription machinery and affinity purification of RNA polymerase II-associated complexes.Methods. 2009; 48: 381-386Crossref PubMed Scopus (73) Google Scholar). Induction for 24–72 h with 3–6 μm ponasterone A (Invitrogen) was used to express the TAP-tagged proteins. Whole cell extracts prepared from induced and non-induced stable EcR-293 cell lines were subjected to purification by the TAP procedure as described previously (20.Jeronimo C. Forget D. Bouchard A. Li Q. Chua G. Poitras C. Thérien C. Bergeron D. Bourassa S. Greenblatt J. Chabot B. Poirier G.G. Hughes T.R. Blanchette M. Price D.H. Coulombe B. Systematic analysis of the protein interaction network for the human transcription machinery reveals the identity of the 7SK capping enzyme.Mol. Cell. 2007; 27: 262-274Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar, 21.Cloutier P. Al-Khoury R. Lavallée-Adam M. Faubert D. Jiang H. Poitras C. Bouchard A. Forget D. Blanchette M. Coulombe B. High-resolution mapping of the protein interaction network for the human transcription machinery and affinity purification of RNA polymerase II-associated complexes.Methods. 2009; 48: 381-386Crossref PubMed Scopus (73) Google Scholar). The TAP eluates were run on SDS gels and stained with silver, and gel slices were excised and digested with trypsin as described previously (20.Jeronimo C. Forget D. Bouchard A. Li Q. Chua G. Poitras C. Thérien C. Bergeron D. Bourassa S. Greenblatt J. Chabot B. Poirier G.G. Hughes T.R. Blanchette M. Price D.H. Coulombe B. Systematic analysis of the protein interaction network for the human transcription machinery reveals the identity of the 7SK capping enzyme.Mol. Cell. 2007; 27: 262-274Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar, 21.Cloutier P. Al-Khoury R. Lavallée-Adam M. Faubert D. Jiang H. Poitras C. Bouchard A. Forget D. Blanchette M. Coulombe B. High-resolution mapping of the protein interaction network for the human transcription machinery and affinity purification of RNA polymerase II-associated complexes.Methods. 2009; 48: 381-386Crossref PubMed Scopus (73) Google Scholar). The resulting tryptic peptides were purified and identified by LC-tandem mass spectrometry (MS/MS) using a microcapillary reversed-phase high pressure liquid chromatography-coupled LTQ-Orbitrap (ThermoElectron) quadrupole ion trap mass spectrometer with a nanospray interface. The peak list files were generated with extract_msn.exe (version February 15, 2005) using the following parameters: minimum mass set to 600 Da, maximum mass set to 6000 Da, no grouping of MS/MS spectra, precursor charge set to auto, and minimum number of fragment ions set to 10. Protein database searching was performed with Mascot 2.2.04 (Matrix Science) against the human NCBInr protein database (version April 2, 2009). There are 10,427,007 sequences in this database. The mass tolerances for precursor and fragment ions were set to 10 ppm and 0.6 Da, respectively. Trypsin was used as the enzyme allowing for up to two missed cleavages. Carbamidomethyl and oxidation of methionine were allowed as variable modifications. A cutoff score of 30 for the first peptide (15 for the additional peptides) for accepting individual MS/MS spectra was established as optimal for the determination of interaction reliability (IR) scores (see supplemental Table S1 for a list of IR scores for individual interactions). IR scores of each interaction between a prey P and a bait B were computed by a predictor using a logistic regression approach as described previously (21.Cloutier P. Al-Khoury R. Lavallée-Adam M. Faubert D. Jiang H. Poitras C. Bouchard A. Forget D. Blanchette M. Coulombe B. High-resolution mapping of the protein interaction network for the human transcription machinery and affinity purification of RNA polymerase II-associated complexes.Methods. 2009; 48: 381-386Crossref PubMed Scopus (73) Google Scholar). The predictor outputs the probability that an interaction between P and B is correct as a function of a weighted sum of the Mascot score of P, the highest Mascot score of all peptides of P, the number of common interacting partners between P and B, the presence of the interaction in reciprocal purifications, the number of baits that found prey P, and the combinations of these. More precisely, the logistic regression is made of five terms representing the five features described with the addition of the squares of four of these features (the reciprocity feature, which is binary, is not squared) and of 10 terms corresponding to the products of the feature values for a total of 19 terms. To train the predictor, a set of 248 positive and 2403 negative examples was derived from gene ontology (22.Ashburner M. Ball C.A. Blake J.A. Botstein D. Butler H. Cherry J.M. Davis A.P. Dolinski K. Dwight S.S. Eppig J.T. Harris M.A. Hill D.P. Issel-Tarver L. Kasarskis A. Lewis S. Matese J.C. Richardson J.E. Ringwald M. Rubin G.M. Sherlock G. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium.Nat. Genet. 2000; 25: 25-29Crossref PubMed Scopus (26642) Google Scholar) with the hypothesis that proteins sharing at least one gene ontology annotation are likely to be interacting and vice versa. Although these sets do not only contain true positive and true negative interactions, they are enriched for a representative subset of correct interaction identifications, which is sufficient for proper training of the predictor. The regression weights of all 19 terms were computed to minimize the cross-entropy error function between the predictions made by the classifier and the labels of all training interactions. To evaluate the accuracy of the predictor, a test set was built from 149 manually identified correct and 54 incorrect interactions based only on strong literature support and not on our interaction data (20.Jeronimo C. Forget D. Bouchard A. Li Q. Chua G. Poitras C. Thérien C. Bergeron D. Bourassa S. Greenblatt J. Chabot B. Poirier G.G. Hughes T.R. Blanchette M. Price D.H. Coulombe B. Systematic analysis of the protein interaction network for the human transcription machinery reveals the identity of the 7SK capping enzyme.Mol. Cell. 2007; 27: 262-274Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar). With the IR score threshold used in this study (>0.7337), we estimated a specificity of about 88%, suggesting an overall rate of false positives lower than 12%. The use of this IR score threshold lowered the sensitivity to 72%, suggesting that 28% of the relevant interactions are left out of the graph in Fig. 1B. In cases where multiple gene products were identified from the same peptide set, all were unambiguously removed from the data set. In the case of multiple isoforms stemming from a unique gene, the isoform with the best sequence coverage was reported. Proteins identified on the basis of a single peptide are listed in supplemental Tables S4 (human) and S5 (yeast), and the individual spectra are comprehensively presented in supplemental Figs. S1 (human) and S2 (yeast). Affinity-purified protein complexes were concentrated by dialysis in buffer F containing 10 mm Hepes, pH 7.9, 100 mm NaCl, 0.1 mm EDTA, 5% glycerol, and 0.5 mm DTT. An aliquot (50 μl) of the concentrated eluate was fractionated on a Superose 6 PC 3.2/30 column (2.4 ml) previously equilibrated in buffer F using the ÄKTA FPLC system (GE Healthcare). The column was run in buffer F at a flow rate of 0.04 ml/min, and 50-μl fractions were collected. Aliquots of each five fractions were pooled, concentrated, and analyzed by Western blot. RPAP4/GPN1 (ON-TARGETplus SMART pool) and control (siCONTROL non-targeting pool) siRNAs (Dharmacon) were transfected into HeLa cells using Oligofectamine (Invitrogen) at an siRNA final concentration of 100 nm. At various time intervals post-transfection, cells were lysed, and RPAP4/GPN1 expression levels were monitored by Western blotting. The following antibodies were used in this study and were obtained from various sources: primary antibody against RPAP4/GPN1 (CIM Antibody Core, Arizona State University, Tempe, AZ), monoclonal antibody against the RNAPII POLR2A/RPB1 subunit (8WG16; Covance), TAP specific anti-calmodulin binding peptide antibody (clone C16T; Upstate), anti-CDK9 antibody (C-20; Santa Cruz Biotechnology), monoclonal anti-β-tubulin antibody (clone TUB 2.1; Sigma), and horseradish peroxidase-conjugated secondary antibody (GE Healthcare). HeLa cells were seeded into 6-well dishes at a starting density of 10,000 cells/well. Double siRNA treatments were on days 0 and 3. Cells were trypsinized on days 1–7 and counted with a hemocytometer. HeLa cells were grown on Lab-Tek (Nunc). Twenty-four hours post-transfection, cells were fixed with 3.7% formaldehyde in PBS and permeabilized with 0.3% Triton X-100 in PBS. DNA was stained with TO-PRO®-3 (Molecular Probes). For immunofluorescence studies, cells were incubated with the first antibody diluted in 5% donkey serum in PBS for 1 h followed by 1 h of incubation with a 1:200 dilution of Alexa Fluor 488- or Cy3-conjugated secondary antibody. Cells were washed with PBS after each step. Cells were mounted using Prolong Gold and SlowFade Gold antifade reagents (Invitrogen). Images were acquired using an LSM 510 or LSM 710 confocal laser scanning microscope and analyzed using LSM Image Browser, version 3.2.0.70 (Zeiss, Toronto, Canada). Yeast cell culture, fixation, conversion to spheroplasts, and permeabilization were performed as previously described (23.Marfatia K.A. Crafton E.B. Green D.M. Corbett A.H. Domain analysis of the Saccharomyces cerevisiae heterogeneous nuclear ribonucleoprotein, Nab2p. Dissecting the requirements for Nab2p-facilitated poly(A) RNA export.J. Biol. Chem. 2003; 278: 6731-6740Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Slides were blocked and hybridized as described above for mammalian cells. Yeast nuclei were treated with 2.5 μg/ml Hoechst 33342 (Molecular Probes) for 30 min. The GFP-RPAP4(Mut) construct was obtained by cloning the full-length RPAP4/GPN1 open reading frame into pGFP2-N1 (PerkinElmer Life Sciences) using the EcoRI and ApaI sites. Transfection of HeLa cells was performed using the Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's protocol. Cells grown in a 150-mm dish were washed with PBS and resuspended in 1 ml of ice-cold lysis buffer (50 mm Tris-HCl, pH 8, 5 mm MgCl2, 0.5% Nonidet P-40, 0.5% sodium deoxycholate, and one tablet of Complete Mini EDTA-free (Roche Applied Science)). The lysate was centrifuged for 2 min at 9400 × g. The supernatant, representing the cytoplasmic fraction, was kept on ice. The pellet was resuspended in lysis buffer and submitted to three freeze-thaw cycles in liquid nitrogen. The lysate was centrifuged for 10 min at 18,400 × g, and the supernatant was kept as the nuclear fraction (24.Lee T.H. Lwu S. Kim J. Pelletier J. Inhibition of Wilms tumor 1 transactivation by bone marrow zinc finger 2, a novel transcriptional repressor.J. Biol. Chem. 2002; 277: 44826-44837Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). The presence of the protein of interest was analyzed by Western blotting. NPA3, the yeast homolog of RPAP4/GPN1, was cloned in plasmid pRS316 (URA3, ampR, CEN6, ARSH4) and pRS415 (LEU2, ampR, CEN6, ARSH4). Plasmid pRS415-NPA3 was used as a template for all mutagenic polymerase chain reactions (PCRs) using phosphorylated primers (details on the primers used in this study are available upon request). Changes in nucleotide sequences were confirmed by DNA sequencing. Yeast strain BY4743–22550 (MATa/α his3Δ1/his3Δ1 leu2Δ0/leu2Δ0 met15Δ0/MET15 ura3Δ0/ura3Δ0 lys2Δ0/LYS2 npa3::kanMX/NPA3) obtained from the yeast knock-out collection (Invitrogen) was transformed with pRS316-NPA3 using a standard protocol (25.Gietz R.D. Woods R.A. Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method.Methods Enzymol. 2002; 350: 87-96Crossref PubMed Scopus (2055) Google Scholar). Transformed cells were sporulated and dissected on yeast peptone-dextrose (YPD) medium. Haploid knocked out strains were selected on yeast complete medium (Yc) lacking uracil containing G418 (200 μg/ml) and counterselected on Yc containing 5-fluoroorotic acid (5FOA) (1 mg/ml). All pRS415-NPA3 plasmids obtained by mutagenesis were transformed into this haploid strain named BCY74 (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 lys2Δ0 npa3::kanMX pRS316-NPA3). Yeast strains and plasmids used in this study are listed in supplemental Table S3. The function of each yeast mutant was assessed by plasmid shuffling on 5FOA lacking leucine so that the only copy of the NPA3 gene is mutated. These strains were cultured to an A600 nm of 1.0, serially diluted (1, 1:5, 1:50, and 1:500), and spotted on Yc-Leu−-Ura− and Yc-Leu− containing 1 mg/ml 5FOA. Each plate was incubated for 3–5 days at 30 °C. Yeast mutants that showed a slow growth phenotype on 5FOA were then spotted on Yc-Leu− with or without 20 μg/ml benomyl with the same serial dilutions. In previous work, we used AP-MS to begin to characterize the network of interactions of the human RNAPII transcription machinery in the soluble fraction of human cell extracts (20.Jeronimo C. Forget D. Bouchard A. Li Q. Chua G. Poitras C. Thérien C. Bergeron D. Bourassa S. Greenblatt J. Chabot B. Poirier G.G. Hughes T.R. Blanchette M. Price D.H. Coulombe B. Systematic analysis of the protein interaction network for the human transcription machinery reveals the identity of the 7SK capping enzyme.Mol. Cell. 2007; 27: 262-274Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar, 26.Jeronimo C. Langelier M.F. Zeghouf M. Cojocaru M. Bergeron D. Baali D. Forget D. Mnaimneh S. Davierwala A.P. Pootoolal J. Chandy M. Canadien V. Beattie B.K. Richards D.P. Workman J.L. Hughes T.R. Greenblatt J. Coulombe B. RPAP1, a novel human RNA polymerase II-associated protein affinity purified with recombinant wild-type and mutated polymerase subunits.Mol. Cell. Biol. 2004; 24: 7043-7058Crossref PubMed Scopus (64) Google Scholar, 27.Krueger B.J. Jeronimo C. Roy B.B. Bouchard A. Barrandon C. Byers S.A. Searcey C.E. Cooper J.J. Bensaude O. Cohen E.A. Coulombe B. Price D.H. LARP7 is a stable component of the 7SK snRNP while P-TEFb, HEXIM1 and hnRNP A1 are reversibly associated.Nucleic Acids Res. 2008; 36: 2219-2229Crossref PubMed Scopus (178) Google Scholar). This work identified a number of previously uncharacterized proteins that associate with known transcription factors to regulate their activity prior to their involvement in active transcription on template chromatin. A subset of these factors was found to be tightly connected to RNAPII in the cellular soluble fraction, suggesting a possible role in RNAPII biogenesis and in regulating the activity of this enzyme before its involvement in active transcription (20.Jeronimo C. Forget D. Bouchard A. Li Q. Chua G. Poitras C. Thérien C. Bergeron D. Bourassa S. Greenblatt J. Chabot B. Poirier G.G. Hughes T.R. Blanchette M. Price D.H. Coulombe B. Systematic analysis of the protein interaction network for the human transcription machinery reveals the identity of the 7SK capping enzyme.Mol. Cell. 2007; 27: 262-274Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar). To further characterize the network of interactions that connects these RPAPs to RNAPII itself, we proceeded to affinity purify additional components of the network present in human 293 cells (see Fig. 1A for a list of TAP-tagged proteins; note that in Fig. 1, A and B, the official gene symbols are used for all proteins, including the RNAPII subunits (POLR2A–POLR2L)). Overall, 28 members of the RNAPII-RPAP network were used in AP-MS experiments. Demonstrative silver-stained gels are shown in supplemental Fig. S3. Fig. 1B illustrates the high density interaction network mapped using this procedure. As we described above, high confidence interactions were selected by applying a computational algorithm that assigns IR scores to each detected interaction according to the strength of the MS score and the local topology of the network (e.g. conservation of the interaction in reciprocal purifications and number of shared partners). Interactions with an IR score over a threshold that minimizes the rate of false positives were selected (20.Jeronimo C. Forget D. Bouchard A. Li Q. Chua G. Poitras C. Thérien C. Bergeron D. Bourassa S. Greenblatt J. Chabot B. Poirier G.G. Hughes T.R. Blanchette M. Price D.H. Coulombe B. Systematic analysis of the protein interaction network for the human transcription machinery reveals the identity of the 7SK capping enzyme.Mol. Cell. 2007; 27: 262-274Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar, 21.Cloutier P. Al-Khoury R. Lavallée-Adam M. Faubert D. Jiang H. Poitras C. Bouchard A. Forget D. Blanchette M. Coulombe B. High-resolution mapping of the protein interaction network for the human transcription machinery and affinity purification of RNA polymerase II-associated complexes.Methods. 2009; 48: 381-386Crossref PubMed Scopus (73) Google Scholar). Fig. 1B shows all the interactions with IR scores higher than 0.7337. Above this threshold, we estimated a specificity of about 88%, suggesting an overall rate of false positives lower than 12%. Of note, the use of an IR score threshold of 0.7337 lowered the sensitivity to 72%, suggesting that 28% of the relevant interactions are left out of the graph in Fig. 1B. The list of interactions obtained using our experimental procedure with their associated IR scores is provided in supplemental Table S1. Examination of the diagram in Fig. 1B reveals that a protein termed RPAP4 (official gene symbol, GPN1) occupies a central position in the RNAPII interaction network by connecting RNAPII to: (i) the CCT complex (28.Leroux M.R. Hartl F.U. Protein folding: versatility of the cytosolic chaperonin TRiC/CCT.Curr. Biol. 2000; 10: R260-R264Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 29.Martín-Benito J. Boskovic J. Gómez-Puertas P. Carrascosa J.L. Simons C.T. Lewis S.A. Bartolini F. Cowan N.J. Valpuesta J.M. Structure of eukaryotic prefoldin and of its complexes with unfolded actin and the cytosolic chaperonin CCT.EMBO J. 2002; 21: 6377-6386Crossref PubMed Scopus (161) Google Scholar); (ii) the RPAP3/R2TP/prefoldin-like complex (30.Zhao R. Kakihara Y. Gribun A. Huen J. Yang G. Khanna M. Costanzo M. Brost R.L. Boone C. Hughes T.R. Yip C.M. Houry W.A. Molecular chaperone Hsp90 stabilizes Pih1/Nop17 to maintain R2TP complex activity that regulates snoRNA accumulation.J. Cell Biol. 2008; 180: 563-578Crossref PubMed Scopus (144) Google Scholar, 31.Sardiu M.E. Cai Y. Jin J. Swanson S.K. Conaway R.C. Conaway J.W. Florens L. Washburn M.P. Probabilistic assembly of human protein interaction networks from label-free quantitative proteomics.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 1454-1459Crossref PubMed Scopus (195) Google Scholar, 32.Gstaiger M. Luke B. Hess D. Oakeley E.J. Wirbelauer C. Blondel M. Vigneron M. Peter M. Krek W. Control of nutrient-sensitive transcription programs by the unconventional prefoldin URI.Science. 2003; 302: 1208-1212Crossref PubMed Scopus (140) Google Scholar), which comprises 11 subunits, including some small molecular weight chaperones called prefoldins; and (iii) a number of additional polypeptides involved in protein assembly and/or folding, including some chaperones. Of note, RPAP4/GPN1 is the only protein in the network connected to the majority of the proteins forming the RNAPII, RPAP3/R2TP/prefoldin-like, and CCT complexes. Affinity purification of tagged NPA3, the yeast homolog of RPAP4/GPN1, confirmed its association with RNAPII and the RPAP3 complex in Saccharomyces cerevisiae and demonstrated the conservation of these interactions in eukaryotes (see supplemental Table S2). To further confirm the association o

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