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

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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 expres- sion (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.
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