HSP90 and Its R2TP/Prefoldin-Like Cochaperone Are Involved in the Cytoplasmic Assembly of RNA Polymerase II

Molecular Cell Article

HSP90 and Its R2TP/Prefoldin-like Cochaperone Are Involved in the Cytoplasmic Assembly of RNA Polymerase II

Se´ verine Boulon,1,5,6 Be´ renge` re Pradet-Balade,2,3,4,5 Ce´ line Verheggen,2,3,4,5 Dorothe´ e Molle,2,3,4,5 Ste´ phanie Boireau,2,3,4 Marya Georgieva,2,3,4 Karim Azzag,2,3,4 Marie-Ce´ cile Robert,2,3,4 Yasmeen Ahmad,1 Henry Neel,2,3 Angus I. Lamond,1 and Edouard Bertrand2,3,4,* 1Wellcome Trust Centre for Gene Regulation and Expression, University of Dundee, Dundee DD1 5EH, UK 2Institut de Ge´ ne´ tique Mole´ culaire de Montpellier - UMR 5535 CNRS, Universite´ Montpellier 2, 34293 Montpellier Cedex 5, France 3Universite´ Montpellier 2, 34095 Montpellier Cedex 5, France 4Universite´ Montpellier 1, 34060 Montpellier Cedex 2, France 5These authors contributed equally to this work 6Present address: Centre de Recherche en Biochimie Macromole´ culaire - UMR 5237, 34294 Montpellier Cedex 5, France *Correspondence: [email protected] DOI 10.1016/j.molcel.2010.08.023

SUMMARY The activities, structures, and subunit composition of the three major RNA polymerases have been characterized in detail RNA polymerases are key multisubunit cellular (Cramer et al., 2008). RNA polymerases I, II, and III are enzymes. Microscopy studies indicated that RNA composed of 14, 12, and 17 subunits, respectively. The two polymerase I assembles near its promoter. However, largest subunits form the catalytic core of the enzyme, while the mechanism by which RNA polymerase II is the others are smaller and generally bind on their surface. The assembled from its 12 subunits remains unclear. three RNA polymerases are related to each other, and this struc- We show here that RNA polymerase II subunits tural similarity is also highlighted by the fact that some subunits are shared by several polymerases, with a few being present in Rpb1 and Rpb3 accumulate in the cytoplasm when all three enzymes. Despite intensive studies on the structure assembly is prevented and that nuclear import of and regulation of RNA polymerases, relatively little is known Rpb1 requires the presence of all subunits. Using about the location and mechanism of their assembly. To date, MS-based quantitative proteomics, we character- this question has been principally addressed by live-cell micros- ized assembly intermediates. These included a cyto- copy techniques that used GFP-tagged subunits in mammalian plasmic complex containing subunits Rpb1 and cells. FRAP (fluorescence recovery after photobleaching) Rpb8 associated with the HSP90 cochaperone studies on RNA polymerase I have indicated that some subunits hSpagh (RPAP3) and the R2TP/Prefoldin-like com- can either assemble or exchange directly at promoters (Dundr plex. Remarkably, HSP90 activity stabilized incom- et al., 2002). Furthermore, the exchange/assembly rate depends pletely assembled Rpb1 in the cytoplasm. Our data on the phase of the cell cycle, indicating that assembly of RNA indicate that RNA polymerase II is built in the cyto- polymerase I in the nucleolus is a way to regulate gene expression (Gorski et al., 2008). Live-cell studies on RNA polymerase plasm and reveal quality-control mechanisms that II promoters have also revealed a rapid exchange of basal and link HSP90 to the nuclear import of fully assembled sequence-specific transcription factors at promoters. In yeast, enzymes. hSpagh also bound the free RPA194 the TATA binding factor TBP and the transcriptional activator subunit of RNA polymerase I, suggesting a general Ace1p were shown to rapidly come on and off the chromatin, role in assembling RNA polymerases. with a residency time in the range of seconds (Karpova et al., 2008; Sprouse et al., 2008). Moreover, the rapidly cycling Ace1p molecules were shown to be responsible for transcrip- INTRODUCTION tional activation. In mammalian cells, glucocorticoid and estra- diol receptors were shown to have similarly high exchange rates RNA polymerases play fundamental roles in the cell. RNA poly- with their target sites on DNA (McNally et al., 2000; Stenoien merases I and III synthesize the noncoding RNAs that form the et al., 2001). More recently, the use of Drosophila salivary glands translational apparatus, and their activity is intimately linked to with polytene chromosomes and the generation of human cell the growth state of the cell (White, 2005). RNA polymerase II lines carrying artificial arrays of reporter genes have allowed synthesizes capped noncoding RNAs as well as all mRNAs. detailed studies of transcription in living cells (Janicki et al., This enzyme is at the heart of gene regulation and is subjected 2004; Yao et al., 2006). In particular, FRAP studies of GFP- to many controls, including at the level of initiation, elongation, tagged subunits of RNA polymerase II were used to define the and termination (Fuda et al., 2009). dynamics of their binding to promoters and the transcription

912 Molecular Cell 39, 912–924, September 24, 2010 ª2010 Elsevier Inc. Molecular Cell HSP90, R2TP, and RNA Polymerase II Assembly

kinetics of this key enzyme. In two studies that used HIV-1 a specific inhibitor of the exportin CRM1 (Fornerod et al., 1997). reporters or natural Drosophila genes, an efficient initiation entry This resulted in the retention of GFP-Rpb3 in the nucleus mode was found (Boireau et al., 2007; Yao et al., 2007). In (Figure 1A), indicating that an active export mechanism removes contrast, a study that used a Tet-inducible promoter in human GFP-Rpb3 from nuclei when Rpb1 is degraded. cells found that a large fraction of the polymerase exchanges Surprisingly, LMB prevented nuclear import of Rpb1 rapidly at promoters, with only a few percent that go into produc- (Figure 1A) and made this subunit insensitive to a-amanitin tive elongation (Darzacq et al., 2007). Given the precedent (Figures 1A and S1). Because a-amanitin specifically promotes example of RNA polymerase I assembly at its promoter, these degradation of the elongating form of RNA polymerase II data led to the suggestion that RNA polymerase II may also (Nguyen et al., 1996), the cytoplasmic accumulation of Rpb1 assemble at its promoter and that this could allow a gene- most likely represents newly synthesized subunits that were specific regulation of its assembly (Darzacq and Singer, 2008; never engaged in transcription elongation. These data suggest Hager et al., 2009). that factors required for Rpb1 nuclear import may be retained Recently, affinity purification of soluble human RNA poly- in the nucleus upon LMB treatment. Since Rpb3 remains nuclear merase II with TAP-tagged subunits identified a number of poly- in these conditions, an intriguing possibility would be that Rpb1 merase-associated factors of unknown function (Jeronimo et al., needs to be incorporated in fully assembled enzymes before 2004, 2007). Four of these factors are homologous to the yeast being imported into the nucleus. R2TP complex (Boulon et al., 2008; Zhao et al., 2005), and further To test this hypothesis, RNA polymerase II assembly was pre- studies suggested that they indeed form a complex that contains vented by inhibiting de novo synthesis of individual subunits with the R2TP factors plus five prefoldin-like proteins (referred to as siRNAs. Depletion of Rpb2, the second largest subunit, pre- R2TP/Prefoldin-like) (Boulon et al., 2008; Cloutier et al., 2009; vented nuclear import of Rpb1 (Figure 1B). The pool of Rpb1 Sardiu et al., 2008). The yeast R2TP protein Tah1 was initially accumulated in the cytoplasm was resistant to a-amanitin, indi- characterized as a cochaperone for HSP90 (Zhao et al., 2005) cating that it corresponds to newly synthesized proteins that and, in agreement, we recently showed that the human Tah1 were never engaged in elongation. Furthermore, analysis with homolog hSpagh (also called RPAP3 or FLJ21908) is an a panel of antibodies that recognize various phosphoisoforms HSP90 cofactor (Boulon et al., 2008). Furthermore, HSP90 and of the CTD heptad-repeat indicated that the cytoplasmic Rpb1 R2TP proteins, including hSpagh, play a key role during the is mostly unphosphorylated (see Supplemental Experimental assembly of snoRNPs (Boulon et al., 2008; Gonzales et al., Procedures and Figure S1). This effect on Rpb1 caused by 2005; King et al., 2001; Zhao et al., 2008), raising the possibility siRNA-mediated depletion of Rpb2 was not an indirect conse- that the association of hSpagh with RNA polymerase II subunits quence of transcription inhibition. Indeed, the transcription inhib- could be involved in the assembly of this enzyme. itors DRB and actinomycin D, which do not trigger subunit In this study, we used a combination of quantitative MS-based degradation (Nguyen et al., 1996), did not induce a cytoplasmic proteomics and fluorescence microscopy to characterize the accumulation of Rpb1 (Figures 1C and S1). These results were mechanism of RNA polymerase II assembly in human cells and extended by examining siRNA-mediated removal of other RNA the role of hSpagh and Hsp90 in this process. polymerase II subunits. Remarkably, depletion of any subunit, including Rpb3, resulted in a similar accumulation of Rpb1 in RESULTS the cytoplasm (Figures 1D and S1, and see below for quantitative measurements of Rpb1 localization). Together with the fact that Unassembled RNA Polymerase II Subunits Accumulate degradation of elongating Rpb1 by a-amanitin triggered nuclear in the Cytoplasm export of Rpb3, these data suggested that assembly of RNA To study assembly of RNA polymerase II, we took advantage of polymerase II occurs in the cytoplasm and is an obligatory a-amanitin. This drug binds the large Rpb1 subunit of human step prior to nuclear import. RNA polymerase II with high affinity. In vitro, this reduces the elongation rate more than 100-fold and makes the polymerase GFP-Tagged Subunits Rpb1 and Rpb3 Have Similar prone to essentially irreversible stalling (Rudd and Luse, 1996). Dynamics at the Transcription Site of an HIV-1 Reporter In vivo, this results in transcriptional arrest and concomitant Gene destruction of subunit Rpb1. The other subunits remain intact, Assembly of RNA polymerase I in the nucleolus was demon- suggesting that a-amanitin may result in enzyme disassembly strated by FRAP using GFP-tagged subunits (Dundr et al., (Nguyen et al., 1996). First, the localization of RNA polymerase II 2002). Indeed, RNA polymerase I subunits have biphasic subunit Rpb3 was analyzed by fluorescence microscopy using recovery curves in the nucleolus, with an initial rapid recovery a U2OS cell line stably expressing a GFP-Rpb3 fusion protein of about 1 min that is specific for each subunit and that repre- known to incorporate into functional RNA polymerase (Boireau sents exchange of their unassembled form. To further substan- et al., 2007 and see below). The GFP-Rpb3 fusion protein nor- tiate that RNA polymerase II is assembled in the cytoplasm, mally accumulates in the nucleus, with only a faint cytoplasmic rather than at promoters, we determined the dynamics of staining (Figure 1A). However, this changed following a-amanitin different polymerase subunits at the transcription site of an addition, with GFP-Rpb3 now accumulating in the cytoplasm. To HIV-1 reporter gene that was tagged with MS2 sites for visualiza- test whether this resulted either from a failure to import newly tion in living cells (Fusco et al., 2003; Boireau et al., 2007) synthesized Rpb3 or instead from export of the nuclear subunit, (Figure 2). To this end, we used the Exo1 cell line, which has inte- cells were treated with both a-amanitin and leptomycin B (LMB), grated many copies of the reporter gene and was shown to

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Figure 1. Disruption of RNA Polymerase II Assembly Induces the Accumulation of Subunits Rpb1 and Rpb3 in the Cytoplasm (A) Cytoplasmic accumulation of subunits Rpb1 and Rpb3 following treatment with LMB or a-amanitin. U2OS cells expressing a GFP-Rpb3 fusion were untreated (NT) or treated for 15 hr with a-amanitin (a-am, 10 mg/ml) and/or leptomycin B (LMB, 15 nM) and processed for immunofluorescence against Rpb1. Each field is 83.75 3 83.75 mm. DAPI stains nuclei. Similar results were obtained after 3 and 6 hr of treatments, although at these time points a-amanitin affects only a fraction of cells (not shown). (B) Depletion of subunit Rpb2 leads to the accumulation of newly synthesized Rpb1 in the cytoplasm. U2OS cells were transfected for 48 hr with siRNA against luciferase (siFFL) or Rpb2 (siRpb2) and treated or not with a-amanitin for 15 hr (10 mg/ml). Cells were processed for immunofluorescence against Rpb1. Each field is 83.75 3 117.25 mm. (C) Inhibition of RNA polymerase II transcription by DRB has no effect on the localization of Rpb1. U2OS cells were treated for 15 hr with DRB (100 mM), and the localization of Rpb1 was determined by immunofluorescence (panels a-Rpb1). FISH against polyA + RNAs was performed in parallel to assess the inhibition of transcription (panel PolyA+). Each field is 67 3 67 mm. (D) Depletion of RNA polymerase II subunits leads to the accumulation of Rpb1 in the cytoplasm. U2OS cells were transfected for 48 hr with siRNA against luciferase (siFFL) or the indicated subunit. Cells were processed for immunofluorescence with an antibody against Rpb1. Each field is 83.75 3 117.25 mm and contains few nondepleted cells that show wild-type nuclear Rpb1 levels. accumulate high levels of RNA polymerase II at its transcription in either the absence or presence of a-amanitin, i.e., when site (Boireau et al., 2007). We previously observed that in this RNA polymerase II enzyme is either mostly nuclear and active system, GFP-tagged subunit Rpb3 was initiating transcription or mostly cytoplasmic and inactive, respectively (see Figure 1). efficiently and did not show a rapid exchange phase during the A triple isotope SILAC labeling scheme was employed first minute of the recovery (Boireau et al., 2007). However, this (Figure 3A) (Trinkle-Mulcahy et al., 2006). Control U2OS cells did not preclude a different behavior for the other subunits were cultivated in medium containing the normal, ‘‘light’’ amino and, in particular, for subunit Rpb1 (Darzacq et al., 2007). To acids, while cells stably expressing GFP-Rpb3 were grown analyze the dynamics of this subunit, we transfected the Exo1 either with ‘‘medium’’ (no treatment) or with ‘‘heavy’’ (a-amanitin cell line with a GFP-tagged Rpb1 construct that carried an treatment) isotope-labeled amino acids. Soluble cell extract was a-amanitin resistance mutation and selected a-amanitin-resis- prepared from each of the light, medium, and heavy cell cultures; tant cell lines to replace the endogenous polymerase by the GFP-Rpb3 and associated partners were affinity purified, and GFP-tagged one. Remarkably, the analysis of GFP-Rpb1 tryptic peptides were analyzed by MS. Intensity ratios for the dynamics by FRAP revealed a recovery rate at the HIV-1 tran- three isotopic forms of each protein were determined using Max- scription site that was not significantly different from the Quant (Cox et al., 2009) and analyzed within Peptracker (Y.A. and GFP-Rpb3 subunit, with an absence of an initial rapid exchange A.I.L., unpublished data). Data were plotted in 2D logarithmic phase. This was consistent with the idea that RNA polymerase II graphs, with the x axis representing enrichment of GFP-Rpb3- was recruited to the HIV-1 promoter as a preformed complex associated proteins in comparison with the control IP rather than as free subunits. (medium/light ratio) and the y axis representing enrichment of GFP-Rpb3-associated proteins in a-amanitin-treated versus Characterization of RNA Polymerase II Assembly untreated cells (heavy/medium ratio) (Figure 3A). Contaminant Intermediates by SILAC Quantitative Proteomics proteins are clustered around the origin, while proteins specifi- Using GFP-Rpb3 as a Bait cally copurified with GFP-Rpb3 in untreated cells appear to the Next, a quantitative MS-based proteomics approach was used right of the graph. Proteins whose specific copurification with to characterize the changes in RNA polymerase II complexes GFP-Rpb3 decreased upon treatment with a-amanitin appear

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speciﬁcity, i.e., RPAP1, RPAP2, GrinL1A, GPN1, GPN2, and GPN3, and the SILAC data show that these factors preferentially associate with Rpb3 subunits that are not assembled in a complete RNA polymerase II enzyme (Figures 3A and 3B and Table S1). Systematic yeast two-hybrid assays also revealed an extensive set of protein-protein interactions (Figure 3B and Table S2). Together, these data indicate the existence of a cytoplasmic RNA polymerase II complex formed by subunits Rpb2, Rpb3, Rpb10, Rpb11, and Rpb12 and identify additional factors associated with this complex.

Large Subunits of RNA Polymerase I and II Associate with the HSP90 Cochaperone hSpagh when Enzyme Assembly Is Prevented Next, a similar SILAC-based proteomics strategy was used to identify factors bound to newly synthesized Rpb1 in cells treated with a-amanitin and LMB (Figure 4A). In this case, an antibody specific for the endogenous Rpb1 subunit was used for immunoaffinity purification (antibody PB-7C2), rather than a GFP fusion protein. Treatment of cells with a-amanitin and LMB prevented the association of most subunits with Rpb1. While Rpb8 was not much affected, all the other subunits were present in Rpb1 complexes in lower amount, from 3-fold to 6-fold (Figure 4A and Table S3). Several additional factors copurified specifically with Rpb1, including some that were previously described (Jeronimo et al., 2007). Remarkably, association of Rpb1 with RPAP2, GPN1, GPN3, GrinL1A, and the R2TP/Prefol- din-like components hSpagh, PFDN2, and UXT was largely unaf- fected by treatment with a-amanitin and LMB, indicating that they all bind Rpb1 prior to its assembly into a complete enzyme. Systematic yeast two-hybrid tests further revealed that UXT interacts with Rpb1 (Table S4) and could thus link it to the R2TP/Prefoldin-like complex (Figure 4D). Figure 2. GFP-Rpb1 and GFP-Rpb3 Associate with an HIV-1 Analysis of protein half-lives using pulsed isotope labeling and Promoter with Similar Dynamics quantitative MS showed no correlation with loss or enrichment in (A) Schematic of the reporter construct used in the assay. The HIV-1 reporter the immunoprecipitates upon a-amanitin treatment (F.M. Bois- plasmid was inserted in multiple tandem copies in the chromatin of U2OS cells. vert and A.I.L., unpublished data), indicating that changes in (B) Recovery of GFP-Rpb1 at the transcription site of the HIV-1 reporter. MS2- complex composition were not due to loss of proteins induced Cherry accumulates at the reporter transcription site (top left panel), together with GFP-Rpb1 (panel ‘‘prebleach’’). Images of GFP-Rpb1 at the indicated by transcriptional shutdown (data not shown). To further rule time points show the regular and slow recovery of the protein at the transcrip- out this possibility, we performed a similar SILAC experiment tion site. in which we analyzed the status of polymerase assembly in cells (C) Comparison of the recovery rate of GFP-Rpb3 and GFP-Rpb1 at the tran- treated with actinomycin D. The time of actinomycin D treatment scription site of the HIV-1 reporter gene. The recovery curves show very similar (7 hr) was chosen to match the time of effective transcriptional profiles for the two proteins. Time is in seconds. arrest during a-amanitin treatment (Figure S2). For this purpose, a triple-labeling SILAC experiment was performed, where we below GFP-Rpb3, while those present above correspond to purified Rpb1 complexes from (1) untreated cells (light amino proteins whose specific copurification with GFP-Rpb3 increased acids), (2) cells treated with actinomycin D (medium amino after treatment. Of the 12 known RNA polymerase II subunits, 11 acids), and (3) cells treated with a-amanitin + LMB (heavy were specifically copurified with GFP-Rpb3, with a high number amino acids). The data were displayed in a 2D graph, with SILAC of peptides identified and quantified (see Table S1). Five of ratios of actinomycin D versus untreated cells on the x axis and these—subunits Rpb4, Rpb5, Rpb7, Rpb8, and Rpb9—clearly ratios of a-amanitin + LMB versus untreated cells on the y axis. dissociated from GFP-Rpb3 in cells treated with a-amanitin, Again, we observed that a-amanitin + LMB induced RNA poly- showing that RNA polymerase II complex is, at least in part, dis- merase II disassembly and association of the Rpb1/Rpb8 dimer assembled after Rpb1 degradation. In agreement, structural with the full R2TP/Prefoldin-like complex (Figure 4B). In contrast, analyses have shown that except for Rpb9, four subunits cited polymerase disassembly was not observed upon treatment with above interact with Rpb3 via subunit Rpb1 (Figure 3B) (Armache actinomycin D. Although an increased association was observed et al., 2003; Bushnell and Kornberg, 2003). Several additional with the R2TP/Prefoldin-like complex, it was clearly less than in factors were found to associate with subunit Rpb3 with high cells treated with a-amanitin + LMB (Figures 4B and 4C). Thus,

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Figure 3. Characterization of Partially Assembled RNA Polymerase II Subcomplexes by Quantitative SILAC Proteomic Analysis (A) Comparison of GFP-Rpb3 complexes in the presence or absence of a-amanitin. Left panel: design of the triple-encoding SILAC GFP-Rpb3 pull-down experiment (see text). Right graph: SILAC results visualized on a 2D logarithmic graph for all proteins identified. SILAC ratios have been normalized so that IP contam- inants are clustered at the origin of the graph. On the x axis, log2(M/L ratio) correlates with the enrichment in GFP-Rpb3 IP versus CTL IP. On the y axis, log2(H/M ratio) correlates with the enrichment in a-amanitin versus nontreated cells. The bait, Rpb3, is spotted in red, and a red line separates the proteins whose interaction with Rpb3 is increased after a-amanitin treatment (above the line) or decreased (below). Variations lower than 2-fold are not considered significant. Only proteins that are significantly enriched in GFP-Rpb3 IP (log[M/L] > 2) are labeled (spotted in orange), plus all R2TP/Prefoldin-like components (spotted in green) and all polymerase subunits (spotted in purple). Proteins that are identified or quantified with less than two peptides are labeled in gray and italic. SILAC ratio values of labeled proteins are listed in Table S1. (B) Model of changes in RNA polymerase II complex and associated factors in untreated cells versus a-amanitin-treated cells, according to SILAC results. The bait is circled in red and the color code is similar to the graph, except that RNA polymerase II subunits that show a decreased association with GFP-Rpb3 upon a-amanitin treatment are displayed in white. Gray lines represent two-hybrid interactions. unassembled Rpb1 is specifically associated with the R2TP/ of Rpb1 with GFP-hSpagh increased upon knockdown of Prefoldin-like complex. Rpb2 (Figure 5B), confirming that hSpagh preferentially binds hSpagh is a central component of the R2TP/Prefoldin-like unassembled Rpb1. Interestingly, hSpagh has also been re- complex that binds HSP90 (Boulon et al., 2008). To determine ported to bind the large subunit of RNA polymerase I, RPA194 possible functions of hSpagh and HSP90 in the biogenesis of (Jeronimo et al., 2007). To test whether this also occurs preferen- RNA polymerase II, we first confirmed the binding of HSP90 tially on the unassembled form of this subunit, cells were treated and hSpagh to Rpb1 by western blotting. Coimmunoprecipita- with siRNA against the second largest subunit of RNA poly- tion (coIP) experiments of endogenous proteins in S100 extracts merase I, RPA135, and GFP-hSpagh immunoprecipitates were revealed that Rpb1 interacted with hSpagh and HSP90, that probed for the presence of RPA194. Indeed, an increased asso- hSpagh was associated with HSP90, and that these interactions ciation of RPA194 with hSpagh was seen upon knockdown of did not depend on HSP90 ATPase activity (Figure S3). Next, we RPA135 (Figure 5C), suggesting that hSpagh preferentially asso- generated a stable U2OS clone expressing a GFP-tagged ciates with free RPA194. version of hSpagh and immunoaffinity-purified GFP-hSpagh. To further characterize the RNA polymerase II complexes This confirmed that Rpb1 was bound to GFP-hSpagh, even after associated with hSpagh, we performed SILAC-based proteomic treating cells with a-amanitin and LMB (Figure 5A). We then analyses using GFP-hSpagh as bait, and we compared protein depleted the Rpb2 subunit with siRNA. Remarkably, interaction complexes from control and a-amanitin-treated cells

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(Figure 5D and Tables S5 and S6). Components of the R2TP/ either a-amanitin + LMB treatment or siRNA-mediated Rpb2 Prefoldin-like complex were readily identified copurifying with depletion (Figures 7A and S1). In addition, quantification of fluo- GFP-hSpagh, but only four RNA polymerase II subunits were rescence microscopy images revealed that GA destabilized found, i.e., Rpb1, Rpb2, Rpb8, and Rpb5. Furthermore, specifically the cytoplasmic form of Rpb1 (Figure 7B). This also a-amanitin-induced destruction of Rpb1 strongly decreased occurred upon siRNA-mediated depletion of every subunit the levels of Rpb2 and Rpb8 copurified with GFP-hSpagh, while except Rpb4, Rpb6, Rpb7, and Rpb9 (Figure 7C). We therefore levels of Rpb5 remained unchanged. Previous SILAC immuno- propose that HSP90 is required to stabilize Rpb1 through most precipitation showed that Rpb1 dissociates from Rpb5 but of the assembly pathway and, in particular, for the joining of remains associated with hSpagh upon treatment with LMB and Rpb1 with Rpb8, Rpb5, and with the Rpb2-Rpb3-Rpb10- a-amanitin (Figure 4A). Altogether, these data suggest that Rpb11-Rpb12 subcomplex. To then test the role of hSpagh in hSpagh associates independently with Rpb5- and Rpb1-con- this process, it was depleted with siRNA. We observed that taining subcomplexes (Figure 5E). removal of hSpagh mimicked the effect of HSP90 inhibition: it destabilized the cytoplasmic Rpb1 that accumulated upon Interaction of hSpagh with Unassembled Subunit Rpb1 depletion of either Rpb2 or Rpb3, but not Rpb6 (Figures 7D Occurs in the Cytoplasm and S5). Interestingly, long-term depletion of hSpagh, for Unassembled subunit Rpb1 accumulates in the cytoplasm, sug- 3 days, resulted in the loss of Rpb1, consistent with hSpagh gesting that its interaction with hSpagh may take place in this having a role in the assembly of RNA polymerase II (Figure 7E). compartment. To test this possibility, we set up a subcellular corecruitment assay (Figure 6)(Darzacq et al., 2006). In these exper- DISCUSSION iments, hSpagh was artificially targeted to distinct subcellular compartments: either to cytoplasmic P-bodies, by fusion to In this study, we have identified a mechanism for the formation of a fragment of p54/RCK (Minshall et al., 2009), or to nucleo- RNA polymerase II from its subunits, which suggests that the plasmic LacO arrays, by fusion to Laci (Darzacq et al., 2006). assembly pathway occurs predominantly, if not entirely, within Subsequent recruitment of interacting partners to either P- the cytoplasm. Analysis of the SILAC quantitative MS data bodies or LacO arrays was then tested to assess their binding permits rigorous and comprehensive evaluation of the dynamic to hSpagh in the cytoplasm or the nucleus, respectively. First, behavior of RNA polymerase II complexes, while fluorescence corecruitment of GFP-HSP90 was tested. In cells that expressed microscopy assays determine their subcellular localization. either p54- or Laci-importin Kpna3 fusion protein as a negative This identified putative assembly intermediates containing control, GFP-HSP90 localized diffusely and did not concentrate a subset of RNA polymerase II subunits and showed that these in P-bodies or at the LacO array (Figure 6A). In contrast, in cells complexes occur predominantly in the cytoplasm. Altogether, that expressed hSpagh fused to either p54 or Laci, GFP-HSP90 we propose a model whereby nuclear entry of RNA polymerase accumulated in P-bodies or at the LacO array, respectively II is restricted to fully assembled enzymes. Previous studies on (Figure 6B). Next, the localization of Rpb1 was analyzed by RNA polymerase I postulated the existence of an assembly immunofluorescence. We found that it was weakly detectable compartment in the nucleolus (Dundr et al., 2002). For RNA poly- in P-bodies containing p54-hSpagh. However, it accumulated merase II, our results indicate that this assembly compartment is there strongly upon depletion of Rpb2, and this was specific, the cytoplasm. This model does not exclude the possibility of because no recruitment was seen in cells expressing p54- additional nuclear reassembly mechanisms for disassembled Kpna3 in the same conditions (Figure 6C). Interestingly, p54 polymerases. However, recycling of RNA polymerase II subunits fusion with truncated versions of hSpagh revealed that different may also be predominantly a cytoplasmic process, rather than domains are responsible for HSP90 and Rpb1 binding (Fig- occurring at polymerase II promoters. In agreement, FRAP ure S4). Moreover, Rpb1 could not be detected at the LacO array experiments using GFP-tagged subunits Rpb1 and Rpb3 show bound by Laci-hSpagh. This indicated that interaction of Rpb1 virtually identical recovery rates at an HIV-1 promoter, with an with hSpagh predominantly occurs in the cytoplasm and sug- absence of a rapid recovery phase during the first minute of gested that the nuclear pool of subunit Rpb1 is mostly present the experiment. This indicates that RNA polymerase II is re- in a completely assembled enzyme that doesn’t interact with cruited as a preformed complex, allowing for an efficient initia- hSpagh. tion entry mode. Our data support the idea that assembly of RNA polymerase II is regulated at the level of the entire cell, HSP90 and hSpagh Are Required to Stabilize rather than in a gene-specific manner, at individual promoters. Unassembled Cytoplasmic Subunit Rpb1 RNA polymerase II is a central enzyme required for the tran- hSpagh is an HSP90 cofactor, and we thus analyzed the effects scription of essentially all pre-mRNAs in eukaryotic cells. The of inhibiting HSP90 activity with geldanamycin (GA). Treatment other main macromolecular machines involved in eukaryotic with GA usually results in the degradation of client proteins gene expression, i.e., ribosomes and spliceosomes, are also (Whitesell et al., 1994), but it had little or no effect on the accumu- composed of multisubunit complexes that are largely assembled lation of Rpb1 following an overnight treatment (Figures 7A and in compartments distinct from their final functional location. 7B). Because hSpagh is more specifically associated with the Thus, ribosomes are mostly assembled within nucleoli but func- unassembled Rpb1 subunit, we tested the effect of GA when tion in mRNA translation in the cytoplasm (Boisvert et al., 2007), combined with polymerase assembly inhibition. Remarkably, while snRNPs mature in the cytoplasm but function to remove GA destabilized unassembled Rpb1 that accumulated upon introns exclusively in the nucleus (Matera et al., 2007).

Molecular Cell 39, 912–924, September 24, 2010 ª2010 Elsevier Inc. 917 Molecular Cell HSP90, R2TP, and RNA Polymerase II Assembly

Figure 4. Unassembled Rpb1 Is Associated with the R2TP/Prefoldin-like Complex (A) Comparison of Rpb1 complexes when RNA polymerase II assembly is normal or inhibited by a-amanitin and LMB. The experimental design is indicated on the left. It is similar to Figure 2, except that cells are treated simultaneously with a-amanitin and LMB (for 15 hr) and that an antibody against endogenous Rpb1 is used. Legend as in Figure 2. (B) Transcriptional shutdown does not result in polymerase disassembly (legend as in A). The experimental design is indicated on the left. Legend is asinFigure 2, except that cells were untreated or treated with a-amanitin + LMB (15 hr) or with actinomycin D (7 hr).

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Separating sites of assembly and function might be beneficial in HSP90 in snoRNP and RNA polymerase assembly may preventing aberrant and potentially disruptive interactions contribute to its established role in cell proliferation and onco- between partially or incorrectly assembled complexes and their genic transformation (Calderwood et al., 2006). substrates. Our proteomics data identified factors that associate specifi- EXPERIMENTAL PROCEDURES cally with the putative RNA polymerase II assembly intermediates. Further studies will be required to characterize the precise Cell Culture, Plasmids, and Antibodies role of these different factors in RNA polymerase II biogenesis. U2OS cells were cultivated in DMEM plus antibiotics and 10% FBS. Cells were transfected with Ca-P coprecipitates or with lipofectamine and plus reagent Interestingly, RPAP2 might be the key import factor of RNA poly- (Invitrogen). Drugs were used at the following concentrations: actinomycin merase II, as it binds the fully assembled enzyme (Jeronimo D, 5 mg/ml; DRB, 100 mM; a-amanitin, 10 mg/ml; GA, 2 mM; LMB, 15 nM, except et al., 2007); it is required for its import and it shuttles in an for the SILAC experiments, where it was reduced to 8 nM. The sequence of LMB-dependent manner (data not shown). In this study, we de- siRNAs is given in the Supplemental Information. ciphered the function of hSpagh, which interacts with unassem- L30-GFP-Rpb3 was described previously (Boireau et al., 2007). The other bled Rpb1, the largest RNA polymerase II subunit, and which is vectors were made using the Gateway technology (Invitrogen), with donor vectors from the human ORFeome (Lamesch et al., 2007) or generated by a cofactor for HSP90. We show that HSP90 stabilizes free Rpb1 PCR. Detailed maps are available upon request. hSpagh TPR1 and TPR2 and that Rpb1 and HSP90 bind separable domains on hSpagh domains encompassed nucleotides 390–690 and 850–1160, respectively. (Figure S4). This suggests that hSpagh delivers unassembled Antibodies against Rpb1 recognized the CTD heptads-repeat (both phos- Rpb1 to HSP90, thereby linking this important chaperone to phorylated and unphosphorylated forms [Figure S1]) and were mouse mono- the assembly mechanism of RNA polymerase II. HSP90 is clonal from Euromedex (PB-7C2) (Souffelweyersheim, France). Antibodies unusual in that it functions at late stages of protein folding, against Rpb5 and RPA194 were mouse monoclonals from Euromedex (PB- with clients in a near-native state, whereas most chaperones 4H7) and Santa Cruz (C-1), respectively. Antibody against Rpb2 was a goat polyclonal from Santa Cruz (S20). GFP-TRAP beads were previously function at earlier stages to facilitate the folding and maturation described and contained a recombinant llama monoclonal and monochain of nascent proteins (Wandinger et al., 2008). HSP90 thus antibody against GFP (Chromotek; Planegg-Martinsried, Germany) (Rothba- appears ideally suited to facilitate assembly of protein uer et al., 2008). Tubulin was detected using a rat antibody from Serotec complexes. Recent systematic studies on HSP90 have sug- (MCA78G) (Colmar, France) and GAPDH using an antibody from Santa Cruz gested a large number of potential client proteins, with diverse (FL-335). functions, but few in vivo roles have been clearly demonstrated (McClellan et al., 2007; Zhao et al., 2005). Here, we provide Two-Hybrid Assays Two-hybrid assays were performed as previously described (Boulon et al., evidence that HSP90 and its cochaperone hSpagh coordinate 2008). the assembly of RNA polymerase II in the cytoplasm. The precise mode of action of HSP90 will require detailed structural data, but Cellular Imaging possible roles could be to maintain Rpb1 in a conformation For analyses in fixed cells, cells were grown on glass coverslips, treated as compatible with assembly, to prevent association with incorrect indicated, and fixed in 4% formaldehyde, 13 PBS for 30 min at RT. After per- partners, and/or to provide a quality-control mechanism to meabilization in 0.5% Triton for 10 min at RT and labeling with the appropriate ensure that the enzyme is properly assembled. Our data also antibodies, slides were mounted in Vectashield. For quantitative imaging of 3 suggest that HSP90 may buffer a transient imbalance in free Rpb1 localization, images were captured with a 40 objective using identical settings. Average gray levels in the cytoplasm and nucleus were measured for subunit stoichiometry, which could, for instance, arise upon a large number of cells (between 80 and 200). Background levels were taken in spontaneous, stochastic fluctuations in gene expression (Raser an area devoid of cells and subtracted to cytoplasmic and nuclear values. and O’Shea, 2005). Signals from individual cells were then averaged and normalized to the nuclear Purification of tagged hSpagh identified not only Rpb1, but signal of untreated cells. also the large subunits of RNA polymerases I and III (Jeronimo For FRAP analyses in live cells, U2OS Exo1 cells were transfected with GFP- et al., 2007 and this study). By western blot analysis, binding of Rpb3 and MS2-Cherry vectors, transcription sites were bleached, and fluorescence recovery was recorded as previously described (Boireau et al., 2007). subunit RPA194 to hSpagh was barely detectable under normal For studies of GFP-Rpb1, U2OS Exo1 cells were transfected with a GFP- conditions but increased markedly upon depletion of subunit tagged a-amanitin-resistant form of subunit Rpb1 (Sugaya et al., 2000), and RPA135 (Figure 5C). This indicates that hSpagh preferentially clones were selected in 5 mg/ml a-amanitin. Individual clones were checked associates with unassembled RPA194, suggesting that HSP90 for expression of the HIV-1 reporter gene and GFP-Rpb1, and suitable cells and hSpagh may be involved in the assembly of all three RNA were then transfected with an MS2-Cherry vector and analyzed by FRAP. polymerases. HSP90 and hSpagh are also involved in the production of snoRNPs (Boulon et al., 2008), and they could Immunoprecipitation Cells were rinsed in PBS and lysed in HNTG for 20 min at 4C. Cellular debris thus assemble the machineries that produce tRNAs, mRNAs, was removed by centrifugation (10 min at 20,000 g), and extracts were incu- and ribosomes. These molecules form the translational appa- bated with appropriate beads (2 hr at 4C). The beads were washed four times ratus, whose activity directly controls production of the cellular in PBS/Triton 0.1% and resuspended in Laemmli buffer. Bound proteins were mass (White, 2005). We therefore propose that the function of analyzed by western blotting. HNTG contained 20 mM HEPES (pH 7.9),

(C) Unassembled Rpb1 is speciﬁcally associated with the R2TP/Prefoldin-like complex. SILAC ratios are from the experiment depicted in (B) and correspond to cells treated with a-amanitin + LMB versus those treated with actinomycin D. The ratios are normalized to that of Rpb1. (D) Model of the complex containing unassembled Rpb1 (legend as in Figure 3).

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Figure 5. hSpagh Preferentially Associates with Unassembled Forms of the Large Subunits of RNA Polymerases I and II (A and B) hSpagh preferentially binds incompletely assembled subunit Rpb1. U2OS cells stably expressing GFP-hSpagh were subjected to GFP-TRAP immunoprecipitation. Inputs and pellets were analyzed by western blots with the indicated antibodies. Cells were untreated (NT) or treated with a-amanitin and LMB

920 Molecular Cell 39, 912–924, September 24, 2010 ª2010 Elsevier Inc. Molecular Cell HSP90, R2TP, and RNA Polymerase II Assembly

Figure 6. hSpagh Interacts with HSP90 and Unassembled Subunit Rpb1 in the Cyto- plasm (A) hSpagh interacts with HSP90 in the nucleus and the cytoplasm. U2OS cells were transfected with the indicated constructs and processed for epifluorescence microscopy. The p54 and Laci constructs were also fused to a red fluorescent protein. Each field is 38 3 38 mm. Insets: zoom on the boxed area (4.3 3 4.3 mm). (B) hSpagh interacts with subunit Rpb1 in the cytoplasm. U2OS cells were transfected with the indicated construct and siRNAs and then processed for immunofluorescence against Rpb1. Legend is as in (A). (C) hSpagh fails to recruit Rpb1 subunit at LacO array in the nucleoplasm. Legend is as in (A) and (B).

150 mM NaCl, 1% Triton, 10% glycerol, 1 mM MgCl2, 1 mM EGTA, and SUPPLEMENTAL INFORMATION protease inhibitors (Roche). Supplemental Information includes Supplemental Experimental Procedures, Proteomic Analyses Supplemental References, ﬁve ﬁgures, and six tables and can be found with The SILAC procedures are described in detail in the Supplemental Information. this article online at doi:10.1016/j.molcel.2010.08.023.

(a-am LMB) (A). Cells were treated with siRNA against Rpb2 (GFP-hSpagh si-Rpb2) or luciferase as control (GFP-hSpagh) (B). The input lanes were loaded with one-tenth of the amount used for the immunoprecipitation. (C) hSpagh associates with unassembled RNA polymerase I subunit RPA194. Cells were treated with siRNA against RPA135 (GFP-hSpagh siRPA135) or luciferase (GFP-hSpagh), subjected to GFP-TRAP puriﬁcation, and analyzed by western blotting with the indicated antibody. The input lanes were loaded with one- tenth the amount of extracts used in the puriﬁcation. (D) Quantitative SILAC proteomic analyses of GFP-hSpagh complexes in cells treated or not with a-amanitin. Left panel shows the experimental design. U2OS cells stably expressing GFP-hSpagh were treated or not with a-amanitin and subjected to SILAC proteomic analysis. Right panel shows SILAC results visualized on a 2D logarithmic graph. x axis: log(M/L ratio) correlates with the enrichment of proteins in GFP-hSpagh IP versus control IP (untreated cells). y axis: log(H/M ratio) correlates with the enrichment of proteins in GFP-hSpagh IP in treated versus untreated cells. (E) Diagram displaying GFP-hSpagh complexes according to SILAC results. Legend is as in Figure 2, except that the thick bars represent direct physical interactions previously demonstrated (Boulon et al., 2008).

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Figure 7. hSpagh and HSP90 Activity Stabilize Unassembled Cytoplasmic Rpb1 Subunit (A) Inhibition of HSP90 activity with GA selectively destabilizes the unassembled Rpb1 subunit. U2OS cells were treated for 43 hr with control siRNAs (siFFL) or with siRNA against Rpb2 (siRpb2) and incubated with GA in the last 15 hr (GA, 2 mM) when indicated. Extracts were prepared in HNTG and analyzed by western blotting with antibodies against Rpb1, Rpb2, and tubulin. (B) Inhibition of HSP90 activity with GA selectively destabilizes cytoplasmic Rpb1 subunit. U2OS cells were treated as above and subjected to immunofluorescence against Rpb1. Each field is 83.75 3 117.25 mm. (C) All stages of polymerase II assembly are not equally sensitive to GA. The amount of subunit Rpb1 in the cytoplasm and nucleus was quantified as follows. Subunit Rpb1 was labeled by immunofluorescence, and images of cells treated as in (B) were taken with identical microscopic settings. Background was removed, and Rpb1 signals in the nucleus and cytoplasm were normalized to the nuclear signal of Rpb1 in untreated cells. Numbers are averages of more than 80 cells; bars are the standard deviations. (D) hSpagh stabilizes unassembled cytoplasmic Rpb1. U2OS cells were treated with the indicated siRNA for 43 hr and processed for immunofluorescence against Rpb1. Each field is 83.75 3 117.25 mm. (E) Destabilization of Rbp1 after long-term depletion of hSpagh. U2OS cells were treated with siRNAs against hSpagh for 48 or 72 hr and analyzed by western blotting with the indicated antibodies.

ACKNOWLEDGMENTS ORFeome 3.1 and U. Rothbauer and H. Leonhardt for the gift of GFP-TRAP beads. We thank R. Bordonne´ for critical reading of the manuscript. A.I.L. is We thank M. Kress for the gift of DsRed2-p54/RCK and the idea of using it to a Wellcome Trust Principal Research Fellow. S. Boulon is a long-term fellow target fusion proteins to P-bodies. We thank M. Vidal for the gift of the human of the Human Frontier Science Program (HFSP), and D.M. had a fellowship

922 Molecular Cell 39, 912–924, September 24, 2010 ª2010 Elsevier Inc. Molecular Cell HSP90, R2TP, and RNA Polymerase II Assembly

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