Gpn3 is required for the nuclear accumulation of RNA polymerase II

Biochimica et Biophysica Acta 1813 (2011) 1708–1716

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Parcs/Gpn3 is required for the nuclear accumulation of RNA polymerase II Mónica R. Calera, Cristina Zamora-Ramos, Minerva G. Araiza-Villanueva, Carlos A. Moreno-Aguilar, Sonia G. Peña-Gómez, Fabiola Castellanos-Terán, Angélica Y. Robledo-Rivera, Roberto Sánchez-Olea ⁎ Instituto de Física, Universidad Autónoma de San Luis Potosí, SLP, México

a r t i c l e

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Article history: Received 6 November 2010 Received in revised form 8 July 2011 Accepted 8 July 2011 Available online 18 July 2011 Keywords: Parcs/Gpn3 nuclear accumulation of RNA polymerase II cell proliferation interference RNA

a b s t r a c t Parcs/Gpn3 is a putative GTPase that is conserved in eukaryotic cells from yeast to humans, suggesting that it plays a fundamental, but still unknown, cellular function. Suppression of Parcs/Gpn3 expression by RNAi completely blocked cell proliferation in MCF-12A cells and other mammary epithelial cell lines. Unexpectedly, Parcs/Gpn3 knockdown had a more modest effect in the proliferation of the tumorigenic MDA-MB-231 and SK-BR3 cells. RNA polymerase II (RNAP II) co-immunoprecipitated with Parcs/Gpn3. Parcs/Gpn3 depletion caused a reduction in overall RNA synthesis in MCF-12A cells but not in MDA-MB-231 cells, demonstrating a role for Parcs/Gpn3 in transcription, and pointing to a defect in RNA synthesis by RNAP II as the possible cause of halted proliferation. The absence of Parcs/Gpn3 in MCF-12A cells caused a dramatic change in the subcellular localization of Rpb1, the largest subunit of RNAP II. As expected, Rpb1 was present only in the nucleus of MCF-12A control cells, whereas in Parcs/Gpn3-depleted MCF-12A cells, Rpb1 was detected exclusively in the cytoplasm. This effect was specific, as histones remained nuclear independently of Parcs/Gpn3. Rpb1 protein levels were markedly increased in Parcs/Gpn3-depleted MCF-12A cells. Interestingly, Rpb1 distribution was only marginally affected after knocking-down Parcs/Gpn3 in MDA-MB-231 cells. In conclusion, we report here, for the first time, that Parcs/Gpn3 plays a critical role in the nuclear accumulation of RNAP II, and we propose that this function explains the relative importance of Parcs/Gpn3 in cell proliferation. Intriguingly, at least some tumorigenic mammary cells have evolved mechanisms that allow them to proliferate in a Parcs/Gpn3-independent manner. © 2011 Elsevier B.V. All rights reserved.

1. Introduction In a previous study we identified a novel protein we named Parcs, for pro-apoptotic protein required for cell survival, by its ability to interact with the oligomerization domain of Apaf-1 [1]. This 33-kDa protein belongs to the Gpn family of GTPases, whose name derives from the presence of a strictly conserved GPN (glycine, proline and asparagine residues) structural motif [2]. The Gpn protein family comprises only three members: Gpn1 (XAB1/MBDin/RPAP4), Gpn2, and Gpn3 (Parcs) [1,3]. Gpn proteins display a high degree of sequence similarity with each other, and all three proteins contain a guanine nucleotide-binding domain. Parcs is highly conserved from yeast to mammals, suggesting that it may serve a fundamental cellular function. In this regard, its ortholog in Saccharomyces cerevisiae is classified as an essential gene [4]. In addition, inactivation of the Parcs ortholog in C. elegans by interference RNA results in embryonic lethality [5–7].

⁎ Corresponding author at: Instituto de Física, Universidad Autónoma de San Luis Potosí, Av. Manuel Nava 6, Zona Universitaria, CP 78290, San Luis Potosí, SLP, México. Tel.: + 52 444 826 2362x110; fax: + 52 444 813 3874. E-mail address: rsanchez@ifisica.uaslp.mx (R. Sánchez-Olea). 0167-4889/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bbamcr.2011.07.005

Parcs/Gpn3 has been reported to participate in the regulation of RNA polymerase II in yeast [8]. RNA polymerase II (RNAP II) is the core component of the complex apparatus responsible of synthesizing all mRNAs in eukaryotic organisms. RNAP II is a large, multi-subunit enzyme, of about 0.5 MDa, which in order to recognize a promoter and initiate RNA synthesis requires additional proteins termed general transcription factors: TFIIA, TFIIB, TFIID, TFIIF, and TFIIH [9–13]. The RNAP II core is composed of at least 12 different proteins encoded by the RPB1 to RPB12 genes, which share extensive conservation among eukaryotic organisms [14–16]. Rpb1, the largest subunit of RNAP II, contains numerous repeats in tandem of a heptapeptide sequence at its carboxy-terminal domain (CTD) [17]. The number of repeats increases with the complexity of the organisms, being 26 and 52 repeats in yeast and human, respectively [18,19]. Phosphorylation of Rpb1 at serine residues on the position 2 and 5 of this heptapeptide is part of a conserved regulatory mechanism of eukaryotic RNA polymerase II function. Parcs/Gpn3 was shown to be a critical protein for cell proliferation in MCF-10A but not in HeLa cells [1]. These two cell lines differ in two aspects: tissue of origin and their tumorigenic nature. MCF-10A cells were derived from breast tissue and are considered very close to normal cells and are, therefore, incapable of forming tumors. HeLa cells, on the other hand, were derived from the cervix and are highly

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tumorigenic. In this study, we investigated the relative importance of Parcs on the proliferation of several cell lines derived from a common origin, the human breast. There is a collection of human breast epithelial cell lines distributed worldwide by ATCC, whose tumorigenic properties have been thoroughly characterized. Based on a previous report documenting that a conditional suppression of the Parcs ortholog in yeast affected the global gene expression pattern in basically the same manner as the inhibition of two different subunits of the RNA polymerase II [8], we investigated, in Parcs-dependent cell lines, the possibility that this multi-subunit enzymatic complex involved in the synthesis of all messenger RNAs is a critical molecular target of Parcs. We demonstrate here that RNAP II co-immunoprecipitates with Parcs/Gpn3. Silencing of Parcs/Gpn3 caused a significantly decrease in cell transcription and a dramatic relocalization of RNP II from the cell nucleus to the cytoplasm. This cytoplasmic Rpb1 is in a hypophosphorylated state, but the total Rpb1 protein levels were significantly increased in the absence of Parcs/Gpn3. Finally, we show that the tumorigenic MDA-MB-231 cells somehow become Parcs independent in all parameters examined: RNAP II is in the cell nucleus, they display normal transcriptional activity and proliferate in the absence of Parcs/Gpn3. 2. Material and methods 2.1. Antibodies and reagents Anti-mouse and anti-rabbit immunoglobulins G conjugated to horseradish peroxidase (HRP), and mouse monoclonal anti-tubulin antibody were obtained from Sigma (St. Louis, MO). Anti-goat immunoglobulin G conjugated to HRP, goat polyclonal anti-Apaf-1 and goat polyclonal anti-Rpb2 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The rabbit polyclonal antibody against Parcs was used as described previously [1]. Mouse monoclonal anti-pan-histone antibody was obtained from Chemicon (Temecula, CA). Rabbit polyclonal anti-phospho-Rpb1 antibodies against Ser-2 and Ser-5 were purchased from Abcam (Cambridge, MA). Mouse monoclonal anti-Rpb1 antibody for total protein (8WG16) was from Covance (Emerville, CA). Chemicals and reagents were obtained from Sigma and, unless otherwise indicated, all tissue culture reagents were from Invitrogen (Carlsbad, CA).

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transfected by the calcium phosphate method and retroviral particles were collected 48 h after transfection. To infect human mammary epithelial cells, the retroviral particles were diluted 1:2 with fresh culture medium and added to the monolayer of cells in the presence of 8 μg/ml polybrene. After removing the viral medium, cells were grown in the presence of puromycin (1.25 ug/ml to 6 μg/ml, depending on the cell line) to select the resistant population of cells. 2.4. Cell proliferation Puromycin-resistant human mammary epithelial cells expressing the indicated Parcs shRNA were trypsinized and counted four days after infection and equal number of cells was plated per well in 6-well plates. Cell proliferation was determined by counting the number of cells four days later. Data point represents the mean ± SD of at least four independent experiments. 2.5. Assessment of RNA synthesis To assess RNA synthesis we employed the method of 5ethynyluridine labeling described by Jao and Salic [22]. MDA-MB468 cells were grown on glass coverslips in complete cell culture medium. 1 mM 5-ethynyluridine (EU, Invitrogen) was added for 1 h at 37 °C. MCF-12A and MDA-MB-231 cells were grown in tissue culture plastic dishes. After EU labeling, cells were washed with phosphate buffered saline solution (PBS) and fixed in 125 mM Pipes (pH 6.8), 1 mM MgCl2, 10 mM EGTA, 0.2% Triton X-100, with 3.7% formaldehyde for 30 min at 25 °C. Fixed cells were rinsed twice with TBS (Tris buffered saline), and stained with a fluorescent azide (Invitrogen). 2.6. Protein extraction Cells were washed with ice-cold PBS and then scraped into lysis buffer consisting of 50 mM Tris (pH 7.4), 1% Triton X-100, 150 mM NaCl, 1 mM MgCl2, 1 mM EDTA, 1 mM DTT and 1 mM PMSF. After incubating cells for 20 min on ice, the lysates were cleared by centrifugation at 13,000 rpm (Sorvall Model 21R) for 10 min at 4 °C. Finally, 2XSDS-containing Laemmli buffer was added to the soluble fraction and proteins were analyzed by immunoblotting (see below).

2.2. Cell culture conditions

2.7. Western blot analysis

All human mammary epithelial cells and HEK 293 T/17 cells were obtained from American Type Culture Collection (Manassas, VA). BT474, MDA-MB-231, MDA-MB-468, SK-BR3 and HEK 293 T/17 cells were grown and maintained in high-glucose Dulbecco´s modified Eagle´s medium (DMEM) supplemented with 10% fetal bovine serum (FBS). MCF-12A was cultured in DMEM-F12 with 5% horse serum, 20 ng/ml EGF, 100 ng/ml cholera toxin, 10 μg/ml insulin and 0.5 μg/ml hydrocortisone [20,21]. All culture medium contained 100 U/ml penicillin and 100 μg/ml streptomycin in a humidified air-CO2 atmosphere at 37 °C.

Samples were separated on SDS- 8 or 10% polyacrylamide gels [23], and then electrophoretically transferred to polyvinylidene difluoride membranes (0.45 μm, Millipore, Billerica, MA) at 25 °C for 30 min. The membranes were incubated in PBST containing 5% nonfat dry milk for 1 h at 25 °C to block nonspecific binding, then probed with the indicated specific antibody. To detect the antigen-bound antibody, the blots were treated with the corresponding secondary antibody conjugated with horseradish peroxidase. Immunoreactivity was detected using the enhanced chemiluminescence system. Immunoblots were quantified by densitometry analysis using the Molecular Imager and Quantity One software (Bio-Rad, Hercules, CA).

2.3. shRNA-mediated depletion of Parcs/Gpn3 2.8. Parcs/Gpn3 immunoprecipitation To stably suppress Parcs expression, we employed pSRP, a selfinactivating retroviral vector, to direct the expression of specific shorthairpin RNAs to suppress Parcs expression. The oligonucleotide sequences of these shRNAs were as follows: g193, GAGGATGATTCTCTGCGAT; g325, GGTCAGATTGAGTTGTACA; and control g239, GCATGGAGTACTTTGCCAA. To suppress Apaf-1 expression we used the sequence we called g2127: GTGACTGCTTCCTCAAACT. Retroviruses were produced as previously described [1]. In brief, retroviruses were produced in HEK 293 T/17 cells with the corresponding plasmid together with gag-pol- and vsv-g-expressing plasmids. Cells were

Adherent cells were washed twice with ice-cold PBS and scraped into 1 ml of lysis buffer (50 mM Tris-base [pH 8.0], 150 mM NaCl, 1% Triton X-100, 1 mM PMSF). Cell lysates were incubated for 20 min on ice and then cleared by centrifugation at 13,000 rpm for 10 min at 4 °C. The soluble fractions were then incubated with a rabbit polyclonal antibody specific for Parcs for 2 h, after which 40 μl of protein A covalently bound to sepharose (Santa Cruz Biotechnology) was added. After rotation for 2 h at 4 0 C, immunocomplexed beads were washed briefly five times with lysis buffer. Beads were heated at

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Parcs/Gpn3 2.9. Immunofluorescence Control or Parcs/Gpn3-depleted MCF-12A or MDA-MB-231 cells were washed twice with PBS and fixed with 4% formaldehyde in PBS (pH 7.4) for 15 min at room temperature. After rinsing the cells with PBS, cells were permeabilized and blocked in one step by incubating them in PBS containing 0.2% Triton X-100 and 10% fetal bovine serum for 15 min at room temperature. Cells were then incubated overnight with either Rpb1 or histones antibody at 4 °C. The Rpb1 mouse monoclonal antibody 8GW16 from Covance was used at a dilution 1:100. The antibody for histones is a pan monoclonal antibody able to detect the histones H1, H2A, H2B, H3 and H4 with equal sensitivity, and it was acquired from Chemicon. After removing the primary antibody, the cells were incubated for three periods of 3 min each in PBS, followed by a 30 min-incubation with Alexa Fluor 568-conjugated anti-mouse secondary antibody acquired from Molecular Probes. Finally, the cells were counterstained with DAPI to visualize nuclei. Images were collected with an Olympus inverted microscope equipped with an evolution MP camera.

3. Results In the initial study describing Parcs/Gpn3, it was reported that whereas the proliferation of MCF-10A cells was completely blocked by suppressing Parcs/Gpn3 expression, HeLa cells could divide normally in the absence of this protein [1]. This finding was really surprising, as Parcs/Gpn3 is an essential protein in yeast and C. elegans (see Introduction). Although MCF-10A and HeLa cells are both of epithelial origin, there is a salient feature that distinguishes them: whereas HeLa cells possess the ability to form tumors, MCF-10A cells lack this property. Thus, it is temping to link this difference with the relative importance of Parcs/Gpn3 in cell proliferation. However, these cell lines were derived from different tissues. Here, we directly investigated the proposal that tumorigenic cells differ from the nontumorigenic ones in their dependence on Parcs/Gpn3 to proliferate. To this end we determined the effect of suppressing Parcs/Gpn3 expression using interference RNA on the proliferative capacity of several human breast epithelial cell lines previously characterized on their tumorigenic capacity. 3.1. Expression of Parcs/Gpn3 in human breast epithelial cell lines We first investigated if Parcs/Gpn3 was expressed in the human breast cell lines examined in this study. Cell extracts were prepared from an equal number of cells per unit of volume, and proteins were separated in SDS-PAGE gels, transferred to PVDF membranes, and probed with a rabbit polyclonal antibody specific for Parcs/Gpn3. The results in Fig. 1 (upper panel) demonstrated that Parcs/Gpn3 is present in all human mammary epithelial cell lines examined. The highest Parcs/Gpn3 protein levels are detected in BT-474 and MCF12A cell lines but Parcs/Gpn3 was also expressed in MDA-MB-231, MDA-MB-468 and SK-BR3 cells.

Tubulin

Fig. 1. Parcs/Gpn3 is expressed in human mammary epithelial cells. Human breast epithelial cells of the indicated cell line were harvested, and total cellular proteins were separated by SDS/PAGE, transferred to PVDF membranes, and immunoblotted with an antibody specific for Parcs/Gpn3 (upper panel) or tubulin (lower panel).

3.2. Effect of silencing Parcs/Gpn3 expression by specific shRNAs on cell proliferation To determine the effect of suppressing Parcs/Gpn3 expression in human mammary epithelial cells on cell proliferation, we first determined the conditions required to effectively decrease Parcs/ Gpn3 protein levels by interference RNA. We employed the retroviral vector pSRP to express Parcs/Gpn3 shRNAs. The packaging of the retroviral particles was carried out in HEK 293 T/17 cells as described in Material and methods. In our initial studies, the Parcs/Gpn3 shRNA g239 was completely ineffective to decrease Parcs/Gpn3 protein levels and we used that construct as a control in all our experiments. Parcs/Gpn3 has previously been demonstrated to be an essential protein for proliferation in normal cells, including primary mouse embryonic fibroblasts and MCF-10A cells [1]. Here, we examined first the effect of suppressing Parcs/Gpn3 expression in MCF-12A cells, another non-tumorigenic cell line very close to normal mammary cells. Parcs/Gpn3 protein levels were almost undetectable in MCF-12A infected with the retrovirus encoding an shRNA (g193) designed to selectively silence Parcs/Gpn3 expression (Fig. 2A, lower panel). Tubulin protein levels were very similar in extracts from cells infected with the retroviral constructs expressing Parcs/Gpn3 shRNAs g239 or g193, confirming that a similar amount of total protein had been loaded in the gels. To assess the effect of Parcs/Gpn3 knockdown on the proliferative capacity of MCF-12A cells, 5 × 10 4 control or Parcs/ Gpn3-depleted cells were seeded per well, and cells were detached with trypsin and counted in a Neubauer chamber four days later. MCF12A cells expressing an empty pSRP retroviral vector, an effective shRNA for Apaf-1, or the ineffective Parcs shRNA g239, increased in number from 5 × 10 4 to around 95 × 10 4. However, cell proliferation was completely inhibited in MCF-12A cells expressing the highly effective Parcs/Gpn3 shRNA g193 (Fig. 2A, upper panel). In fact, Parcs/ Gpn3 knockdown caused a decrease in cell number from 5 to 2.3 × 10 4 in the same period. This result demonstrates that MCF-12A cells depend completely on the presence of Parcs to proliferate. Next we investigated the importance of Parcs/Gpn3 in the proliferation of other breast-derived epithelial cell lines. We began our analysis with the tumorigenic cell line MDA-MB-231. Parcs/Gpn3 is expressed in MDA-MB-231 cells and its protein levels were successfully reduced by the Parcs/Gpn3 shRNA g193, and by a second Parcs/Gpn3 shRNA (g325) targeting a different region of Parcs/Gpn3 mRNA (Fig. 2B, lower panel). To assess the proliferative capacity of MDA-MB-231 cells, 20 × 10 4 cells were seeded per well in 6-well

Fig. 2. Parcs/Gpn3 is essential for cell proliferation in some, but not all, human breast epithelial cells. An equal number of cells expressing the indicated Parcs/Gpn3 shRNAs (either the non-effective, control Parcs/Gpn3 shRNA g239 or the highly effective shRNAs to downregulate Parcs/Gpn3, g193 or g325) were seeded four days after infection with the retroviral particles into 6-well tissue culture plates and grown for four more days before counting the total number of cells (A to E). The initial number of cells plated per well was adjusted for the different cell lines in order to have sub-confluent cultures during the whole evaluation period. Results are the average ± S.D. (n = at least 3 independent experiments). Parcs/Gpn3 and tubulin protein levels at the beginning of the proliferation experiment are shown at the bottom of each panel. Human epithelial breast cell lines examined are: A) MCF-12A, B) MDA-MB-231, C) SK-BR3, D) BT-474, E) MDA-MB-468. In A (MCF-12A cells) we include two more shRNAs as controls, pSRP empty vector and an shRNA effective to knockdown Apaf-1. The ratio for Parcs/Gpn3 protein levels between shRNA g193 or shRNA g325 and shRNA g239 is indicated below the corresponding panels and it was calculated after normalizing for protein loading using tubulin. For Apaf-1 the ratio between g2127 and the empty pSRP vector is shown.

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Fig. 3. RNA polymerase II co-immunoprecipitates with Parcs/Gpn3. MDA-MB-468 human mammary epithelial cells were lysed, and soluble cell extracts subjected to immunoprecipitation with a Parcs/Gpn3 specific rabbit polyclonal antibody. Proteins in the immunoprecipitate were separated by SDS-PAGE and transferred to a PVDF membrane. Western blot analysis was performed with anti-Rpb1 (upper panel) or Rpb2 (lower panel) antibodies as described in Material and methods.

plates and cultured for four days. Cells infected with the control Parcs/ Gpn3 shRNA g239 increased in number from 20 to 103.8 × 10 4 cells. Interestingly, MDA-MB-231 cells expressing the Parcs/Gpn3 shRNAs g193 or g325 increased from 20 to 80.2 and 58.9 × 10 4, respectively (Fig. 2B). Thus, both Parcs/Gpn3 shRNAs caused a rather moderate decrease in cell proliferation. We then extended our analysis to the tumorigenic SK-BR3 cell line. Parcs/Gpn3 shRNAs, g193 and g325, were highly effective to suppress Parcs/Gpn3 expression, as Parcs/ Gpn3 protein levels were almost undetectable (Fig. 2C, lower panel). SK-BR3 cells expressing the Parcs/Gpn3 shRNA control g239 increased from 20 to 69.9 × 10 4 cells during the proliferation assay. SK-BR3 cells expressing the highly effective Parcs/Gpn3 shRNAs g193 or g325 increased from 20 to 47.6 or 48.8 × 10 4 cells, respectively (Fig. 2C). Thus, the importance of Parcs/Gpn3 in cell proliferation was significantly different in the tumorigenic cells MDA-MB-231 or SKBR3 and the non-tumorigenic MCF-12A cells. We then, extended our analysis to two more tumorigenic human breast cancer cell lines: BT474 and MDA-MB-468 cells. Cell proliferation was completely inhibited in the absence of Parcs/Gpn3 in both tumorigenic cell lines, BT-474 (Fig. 2D) and MDA-MB-468 (Fig. 2E). Both Parcs/Gpn3 shRNAs, g193 and g325, were able to successfully decrease Parcs/ Gpn3 protein levels in BT-474 cells (Fig. 2D, lower panel). Control (Parcs/Gpn3 shRNA g239) BT-474 cells increased from 20 to 57.7 × 10 4 during the proliferation test period but cells expressing the Parcs/Gpn3 shRNA g193 o g325 did not proliferate at all, as the initial value of 20 actually decreased to 12.55 or 18.25 × 10 4, respectively (Fig. 2D). We obtained similar results with MDA-MB468 cells. Parcs/Gpn3 protein levels were dramatically reduced in MDA-MB-468 cells expressing the Parcs/Gpn3 shRNA g193 (Fig. 2E, lower panel). MDA-MB-468 cells expressing the ineffective Parcs/ Gpn3 shRNA control g239, increased from 15 to 45.2 × 10 4, whereas cells expressing the Parcs/Gpn3 shRNA g193 in fact decreased from 15 to 10.3 × 10 4. These results demonstrate that although Parcs/Gpn3 expression was successfully suppressed in all the tumorigenic human mammary epithelial cell lines examined, some cells still proliferate in its absence. Perhaps, these cells have developed alternative mechanisms to proliferate. 3.3. RNA polymerase II co-immunoprecipitates with Parcs/Gpn3 As mentioned above, Parcs/Gpn3 belongs to the Gpn protein family. Gpn1, another member of this family, was recently identified by mass spectrometry as a protein that associates with RNA polymerase II (RNAP II) [8], and given the name of RPAP4. Based on

this finding, we explored the possibility that Parcs/Gpn3 could also associate with RNAP II. Parcs/Gpn3 was immunoprecipitated from soluble cell extracts with a rabbit polyclonal antibody. Proteins in this immunoprecipitate were separated by SDS-PAGE, transferred to PVDF membrane and blotted with an antibody specific for Rpb1. The signal corresponding to Rpb1 was clearly present in the Parcs immunoprecipitate (Fig. 3), demonstrating that these proteins are indeed part of a single protein complex. Next, we examined if other subunits of RNAP II were also present in the Parcs immunoprecipitate. Rpb2, the second largest subunit of RNAP II, was also present in this material (Fig. 3). The association of Rpb1 and Rpb2 with Parcs was specific, as they were not detected in the control immunoprecipitate (Fig. 3). These results clearly demonstrate that Parcs has the ability to physically associate with RNAP II. 3.4. Silencing of Parcs/Gpn3 decreased transcriptional activity in cell lines whose proliferation was Parcs-dependent but not in those that proliferate independently of Parcs/Gpn3 A previous report demonstrated that silencing the gene encoding the yeast ortholog of Parcs/Gpn3, YLR243W, in a yeast strain with conditional promoters, affected gene expression in an identical manner to suppressing two different proteins associated with RNA polymerase II [8]. As Parcs/Gpn3 is a highly conserved protein during evolution, we hypothesized that its function in mammalian cells must be very similar to the one observed in yeast. Next, we investigated if the silencing of Parcs/Gpn3 resulted in an overall inhibition in transcription in mammalian cells. MDA-MB-468 cells were infected with retroviral particles encoding Parcs/Gpn3 shRNA g239 or g193, selected with puromycin and plated on glass coverslips. To qualitatively assess RNA synthesis, we employed a recently described method that relies in the incorporation of the uridine analog 5ethynyluridine (see Material and methods) into RNA transcripts, followed by the covalent labeling of the incorporated EU with a fluorofore [22]. We found that MDA-MB-468 cells infected with the Parcs/Gpn3 shRNA control g239 display a very strong signal, indicating a high transcriptional activity (Fig. 4A). This signal was clearly diminished in MDA-MB-468 cells expressing the Parcs/Gpn3 shRNA g193 (Fig. 4A). These results demonstrated for the first time that mammalian cells deficient in Parcs/Gpn3 expression had an important deficiency in transcriptional activity, and we propose that this decrease in transcriptional activity is causally linked to the inhibition in cell proliferation we described above. To test this proposal, we assessed the effect of depleting Parcs/Gpn3 on the transcriptional activity of other two cell lines, MCF-12A and MDA-MB231 cells, whose proliferation is dependent and independent of Parcs/ Gpn3, respectively. The transcriptional activity of MCF-12A (Fig. 4C), but not MDA-MB-231 (Fig. 4E), was markedly reduced by Parcs/Gpn3 depletion. These results confirm that Parcs/Gpn3 is required for an optimal transcriptional activity in those cells that proliferate in a Parcs/Gpn3-dependent manner, but not in those cells that proliferate independently of Parcs/Gpn3. In any case, it is clear that the molecular mechanism controlled by Parcs/Gpn3 in the cell is at the level of transcription, very likely, by RNAP II (see below). RNAP II can exist in several forms based on the phosphorylation state of Rpb1, the largest subunit of the enzyme. One form is nominated IIA, and corresponds to the transcription initiating form of the enzyme that contains a non-phosphorylated carboxy-terminal domain (CTD), an unusually long extension appended to the carboxyterminus of Rpb1. The other form is called II0, which is the fraction of the enzyme engaged in transcription elongation, and contains a highly phosphorylated CTD. This phosphorylation occurs principally on Ser2 and Ser5 [24,25] of the repeated heptapeptide. The phosphorylation status of Rpb1 is an event that is often used as a marker to distinguish specific phases of the polymerase II cycle of activity. While phosphorylation on Ser2 is a late event during transcription

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Fig. 4. The suppression of Parcs expression by RNAi caused a global decrease in transcriptional activity. RNA synthesis was assessed as described in Materials and methods. A decrease in the fluorescence signal corresponds to a reduction in transcriptional activity. shRNA g239 corresponds to the control condition, and the shRNA g193 indicates the Parcs/Gpn3 knockdown cells. A) RNA synthesis in MDA-MB-468. B) Western blot analysis of MDA-MB-468 total cell extracts from panel A with the antibodies for the proteins indicated on the left side of the panels. The ratio of PSer5 Rpb1/Rpb1 is indicated below the top panel. C) RNA synthesis in MCF-12A cells. D) Western blot analysis of MCF-12A total cell extracts from panel C with the antibodies for the proteins indicated on the left side of the panels. The ratio of PSer2 Rpb1/Rpb1 is indicated below the top panel. E) RNA synthesis in MDA-MB-231 cells. Parcs/Gpn1 protein levels are also indicated.

elongation, phosphorylation on Ser5 of the CTD occurs early in the transcriptional cycle and is linked to RNA polymerase II release from the promoter. As a first approximation to investigate the regulatory mechanism of RNA polymerase II by Parcs/Gpn3, we evaluated the effect of suppressing Parcs/Gpn3 expression on the phosphorylation state of Rpb1-CTD on Ser5. Total cell lysates of MDA-MB-468 cells expressing the Parcs/Gpn3 shRNA control g239 or g193 were prepared, proteins were separated in SDS-PAGE, transferred to PVDF membrane and probed with a specific antibody for phospho-Ser5 on the CTD. Analysis by western blot demonstrated that the signal corresponding to phospho-Ser5 Rpb1 was very robust in control cells (Fig. 4B) and that this signal was markedly reduced in cells with suppressed Parcs/Gpn3 expression (Fig. 4B). The decrease in phospho-Ser5 is not caused by a reduction in total Rpb1 total protein levels. Interestingly, cells lacking Parcs/Gpn3 showed a tendency to increase the total Rpb1 protein levels (Fig. 4B, middle panel), probably

as a compensatory mechanism to sustain the transcriptional needs of the cell. A clear reduction in the phosphorylation of Ser2 in the Rpb1 CTD was observed after depleting Parcs/Gpn3 in MCF-12A cells (Fig. 4D). The absence of Parcs/Gpn3 resulted in an even more pronounced increase in Rpb1 protein levels in MCF-12A cells (Fig. 4D). Altogether, these results demonstrate that Parcs/Gpn3 is a critical regulator of the transcriptional activity of RNAP II. 3.5. Parcs/Gpn3 is required for the nuclear localization of RNA polymerase II It is extremely surprising that there is abundant functional and structural information on RNAP II, but absolutely nothing is known on the mechanism that allows the enzyme to enter the cell nucleus. It is clear that none of the 12 constitutive subunits of RNAP II contains a classical, or otherwise recognizable nuclear localization signal.

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A Rpb1

DAPI

MCF-12A Merge

Parcs/ Gpn3 shRNA Control (g239)

Parcs/ Gpn3 shRNA (g193)

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B Histones

DAPI

MCF-12A Merge

Parcs/ Gpn3 shRNA Control (g239)

Parcs/ Gpn3 shRNA (g193)

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C

Rpb1

DAPI

MDA-MB-231 Merge

Parcs/ Gpn3 shRNA Control (g239)

Parcs/ Gpn3 shRNA (g193)

40X Fig. 5. Parcs/Gpn3 depletion causes an inhibition in the nuclear localization of RNAP II. Parcs/Gpn3 was depleted and the sub-cellular localization of the Rpb1 subunit of RNAP II was examined by immunofluorescence. In A and C, the left column corresponds to the Rpb1 signal, the middle column to the nuclear marker DAPI, and the right column to a merge of these two signals. In B, the left column corresponds to the histones, the middle column to the nuclear marker DAPI, and the right column to a merge of these two signals. A) Subcellular localization of Rpb1 in control (shRNA g239, upper panel) or Parcs/Gpn3 knockdown (shRNA g193, lower panel) MCF-12A cells. B) Sub-cellular localization of histones. The antibody used can detect all five histones, H1, H2A, H2B, H3 and H4, in control (shRNA g239, upper panel) or Parcs/Gpn3 knockdown (shRNA g193, lower panel) MCF-12A cells. C) Sub-cellular localization of Rpb1 in control (shRNA g239, upper panel) or Parcs/Gpn3 knockdown (shRNA g193, lower panel) MDA-MB-231 cells.

However, somehow, RNAP II ends up in the cell nucleus, where its DNA substrate is located. While our work was in revision, the first protein needed for the nuclear accumulation of RNAP II [26] was

described and termed RPAP4. Surprisingly, this protein turned out to be Gpn1, a protein that belongs to the same protein family as Parcs/Gpn3. There is a high degree of similarity at the amino acid

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sequence level and it is likely that they could both play the same function. An involvement of Parcs/Gpn3 in the nuclear accumulation of RNAP II could explain all our findings: a physical association of Parcs/Gpn3 with RNAP II, a clear inhibition in transcriptional activity after Parcs/Gpn3 depletion, as well as a reduction in the phosphorylation state of the Rpb1 subunit of RNAP II. Next, we performed experiments to examine the possibility that Parcs/Gpn3 could function in a similar manner to Gpn1/RPAP4 and that it would be, therefore, involved in the nuclear localization of RNAP II. Thus, we analyzed the effect of Parcs/Gpn3 depletion on the cellular localization of Rpb1. For these studies we employed two cells lines: MCF-12A cells, which depend entirely on Parcs/Gpn3 to proliferate, and MDAMB-231 cells, which proliferate independently of Parcs. As expected, in both control MCF-12A and MDA-MB-231 cells, Rpb1 was detected exclusively in the cell nucleus (Fig. 5A and 5C, respectively). However, Rpb1 distribution was very different in these two cell lines in the absence of Parcs. While in the this condition, Rpb1 could still be detected only in the nucleus of MDA-MB-231 cells, in the MCF-12A cells Parcs-depletion caused an impressive change in Rpb1 distribution, and Rpb1 was exclusively detected in the cytoplasm of the cells (Fig. 5A). This relocation of Rpb1 was not the result of a non-specific effect in the nuclear transport systems, or a general loss of nuclear membrane selectivity, as histones were nuclear in control and Parcs/ Gpn3 knocked-down cells (Fig. 5B). This result points to a specific effect of Parcs/Gpn3 depletion on the sub-cellular distribution of RNAP II. These results can be summarized in two critical findings: first and most importantly, that Parcs/Gpn3 plays a critical role in the nuclear accumulation of Rpb1 in mammalian cells, and second, that the relative importance of Parcs/Gpn3 in cell proliferation is directly related to the importance of this protein in the nuclear accumulation of RNAP II. This result is also consistent with the sustained transcriptional activity of Parcs/Gpn3 knockdown MDA-MB-231 cells (see Fig. 4E). It will be of great interest to determine why Parcs/Gpn3 does not participate in the nuclear accumulation of RNAP II in a few cell lines, especially when, until now, all these cell lines have in common is that they all are tumorigenic in nature. 4. Discussion In this report we document that the recently isolated protein Parcs/Gpn3 [1] is widely expressed in human breast epithelial cell lines and provide experimental evidence that the suppression of Parcs/Gpn3 expression by interference RNA leads to a complete inhibition of cell proliferation in MCF-12A and other mammary epithelial cell lines, including BT-474 and MDA-MB-468 cells. Intriguingly, cell proliferation was more modestly affected by Parcs/ Gpn3 inhibition in MDA-MB-231 and SK-BR3 cells. To investigate the molecular mechanisms regulated by Parcs/Gpn3, we first demonstrated that endogenous Parcs/Gpn3 is in the same protein complex as RNAP II, and that the overall transcriptional activity is reduced in Parcs/Gpn3 depleted cells. We succeeded in identifying the molecular mechanism controlled by Parcs/Gpn3, as we demonstrated that in the absence of Parcs/Gpn3, RNAP II fails to be nuclear and localizes completely to the cytoplasm of the cell. In addition, Rpb1 protein levels are markedly increased in cells with suppressed expression of Parcs/Gpn3. Although Parcs is an essential protein to sustain life in all organisms examined (see introduction) there is little informational cues on the molecular mechanisms regulated by this protein in the cell. Since its original description [1], it was clear that Parcs/Gpn3 was fundamental for cell proliferation in MCF-10A cells, although HeLa cells proliferated normally in the absence of this protein. Proliferation in the absence of Parcs may be a peculiarity of HeLa cells or it may be a shared property with other cells. Here we explore the importance of Parcs/Gpn3 in the proliferation of several human breast epithelial cell lines, including the non-tumorigenic MCF-12A cells and the tumor-

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igenic cells lines BT-474, MDA-MB-231, MDA-MB-468 and SK-BR3 cells. MCF-12A cells and two of the tumorigenic cell lines, BT-474 and MDA-MB-468, displayed a complete dependence on Parcs/Gpn3 to proliferate, but cell proliferation of two tumorigenic cell lines, MDAMB-231 and SK-BR3, was much less affected by Parcs/Gpn3 depletion. It is tempting to speculate that there could be a relationship between the tumorigenic properties of the cells and their ability to proliferate in the absence of Parcs/Gpn3, as all cell lines that proliferate without Parcs/Gpn3 were tumorigenic cells: HeLa [1], MDA-MB-231 and SKBR3 (this work). It is likely that during the transformation process leading to tumorigenesis, at least a subgroup of cells acquire changes that endow them with mechanisms to cope or to compensate for the absence of Parcs/Gpn3. We would not anticipate there would be many normal cell types with the capacity to proliferate in the absence of Parcs/Gpn3. Inhibition of Parcs/Gpn3 in mouse embryonic fibroblasts isolated from a knock-out mouse engineered to conditionally inactivate a floxed Parcs/Gpn3 allele with Cre recombinase, caused a complete arrest in cell proliferation [1]. In addition, the constitutive Parcs/Gpn3 knock-out mouse dies very early in development (Sanchez-Olea, R. and J. Yuan, unpublished results), again supporting the idea that Parcs/Gpn3 plays an essential role in the cell. There has been some evidence for a functional link between Parcs/ Gpn3 and RNAP II in yeast. Conditional silencing of the Parcs/Gpn3 ortholog in yeast (YLR243W) inhibited gene expression in the same manner as suppressing either Rpb11 or Rpb50, two individual subunits of RNAP II [8]. We demonstrated here for the first time that Parcs/Gpn3 is also needed for transcriptional activity of RNA polymerase II in mammalian cells. Our findings that the suppression of Parcs/Gpn3 expression reduces overall transcriptional activity in MDA-MB-468 and MCF-12A cells provide evidence that Parcs/Gpn3 is involved in regulating transcription in both, yeast and human cells. An involvement of Parcs/Gpn3 in a basic cellular function such as transcription would explain the conservation of this protein through evolution. Notably, our data clearly demonstrate that the mechanism by which Parcs/Gpn3 is necessary for optimal transcriptional activity is through the correct sub-cellular localization RNAP II. We have clearly demonstrated that in the absence of Parcs/Gpn3 the Rpb1 subunit of RNAP II is in the wrong cellular compartment: the cytoplasm. Thus, Parcs/Gpn3 joins the selected group of proteins involved in the nuclear accumulation of RNAP II, which until our report included only two very recently described proteins, Gpn1/ RPAP4 [26] and Iwr1 [27]. Parcs/Gpn3 may be participating directly in the process of RNAP II nuclear import, or in the nuclear retention of RNAP II. It is possible that Parcs/Gpn3 could theoretically participate in RNAP II assembly, as the inhibition of this process by depleting any subunit of RNAP II with specific siRNAs also results in a cytoplasmic accumulation of Rpb1 [28]. Parcs/Gpn3 was described as a protein that directly binds to, and is needed for optimal activity of Apaf-1 [1]. A role for Parcs/Gpn3 as a chaperone for Apaf-1 is the best model to explain why this protein is important for optimal Apaf-1 function [1]. So, an attractive, but totally speculative model, would be that Parcs/ Gpn3 could function as a chaperone for RNAP II assembly in the cytoplasm, a step apparently crucial for RNAP II nuclear import [28]. The mechanisms controlling the assembly, entry and retention of RNAP II in the cell nucleus have finally started to be revealed. These research topics will certainly be very active and will yield exciting fruits in the near future. Acknowledgments From RSO: With love, admiration and gratitude to my exemplary mentor, Dr. Herminia Pasantes. We thank the President of the Universidad Autónoma de San Luis Potosí (UASLP), Mario García Valdez, as well as to the Academic Secretary, Luz María Nieto Caraveo, for their generous and unconditional support to the creation and development of the Undergraduate Program in Biophysics at the

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UASLP, and the Laboratory of Cellular and Molecular Biology at the Physics Institute. This project was supported by grant CONACYT CIENCIA BASICA number 83751, grant FMSLP-2008-CO1-86772 from FOMIX-SLP, and Institutional funds from UASLP no. C09-FRC-09-17.47 and C10-FRC07-30.31 to Roberto Sánchez-Olea. This project was also supported by grant CONACYT CIENCIA BASICA number 106139, to Mónica R. Calera. References [1] R. Sanchez-Olea, S. Ortiz, O. Barreto, Q. Yang, C.J. Xu, H. Zhu, J. Yuan, Parcs is a dual regulator of cell proliferation and apaf-1 function, J. Biol. Chem. 283 (2008) 24400–24405. [2] S. Gras, V. Chaumont, B. Fernandez, P. Carpentier, F. Charrier-Savournin, S. Schmitt, C. Pineau, D. Flament, A. Hecker, P. Forterre, J. Armengaud, D. Housset, Structural insights into a new homodimeric self-activated GTPase family, EMBO Rep. 8 (2007) 569–575. [3] M. Nitta, M. Saijo, N. Kodo, T. Matsuda, Y. Nakatsu, H. Tamai, K. Tanaka, A novel cytoplasmic GTPase XAB1 interacts with DNA repair protein XPA, Nucleic Acids Res. 28 (2000) 4212–4218. [4] G. Giaever, A.M. Chu, L. Ni, C. Connelly, L. Riles, S. Veronneau, S. Dow, A. LucauDanila, K. Anderson, B. Andre, A.P. Arkin, A. Astromoff, M. El-Bakkoury, R. Bangham, R. Benito, S. Brachat, S. Campanaro, M. Curtiss, K. Davis, A. Deutschbauer, K.D. Entian, P. Flaherty, F. Foury, D.J. Garfinkel, M. Gerstein, D. Gotte, U. Guldener, J.H. Hegemann, S. Hempel, Z. Herman, D.F. Jaramillo, D.E. Kelly, S.L. Kelly, P. Kotter, D. LaBonte, D.C. Lamb, N. Lan, H. Liang, H. Liao, L. Liu, C. Luo, M. Lussier, R. Mao, P. Menard, S.L. Ooi, J.L. Revuelta, C.J. Roberts, M. Rose, P. RossMacdonald, B. Scherens, G. Schimmack, B. Shafer, D.D. Shoemaker, S. SookhaiMahadeo, R.K. Storms, J.N. Strathern, G. Valle, M. Voet, G. Volckaert, C.Y. Wang, T.R. Ward, J. Wilhelmy, E.A. Winzeler, Y. Yang, G. Yen, E. Youngman, K. Yu, H. Bussey, J.D. Boeke, M. Snyder, P. Philippsen, R.W. Davis, M. Johnston, Functional profiling of the Saccharomyces cerevisiae genome, Nature 418 (2002) 387–391. [5] P. Gonczy, C. Echeverri, K. Oegema, A. Coulson, S.J. Jones, R.R. Copley, J. Duperon, J. Oegema, M. Brehm, E. Cassin, E. Hannak, M. Kirkham, S. Pichler, K. Flohrs, A. Goessen, S. Leidel, A.M. Alleaume, C. Martin, N. Ozlu, P. Bork, A.A. Hyman, Functional genomic analysis of cell division in C. elegans using RNAi of genes on chromosome III, Nature 408 (2000) 331–336. [6] J.F. Rual, J. Ceron, J. Koreth, T. Hao, A.S. Nicot, T. Hirozane-Kishikawa, J. Vandenhaute, S.H. Orkin, D.E. Hill, S. van den Heuvel, M. Vidal, Toward improving Caenorhabditis elegans phenome mapping with an ORFeome-based RNAi library, Genome Res. 14 (2004) 2162–2168. [7] B. Sonnichsen, L.B. Koski, A. Walsh, P. Marschall, B. Neumann, M. Brehm, A.M. Alleaume, J. Artelt, P. Bettencourt, E. Cassin, M. Hewitson, C. Holz, M. Khan, S. Lazik, C. Martin, B. Nitzsche, M. Ruer, J. Stamford, M. Winzi, R. Heinkel, M. Roder, J. Finell, H. Hantsch, S.J. Jones, M. Jones, F. Piano, K.C. Gunsalus, K. Oegema, P. Gonczy, A. Coulson, A.A. Hyman, C.J. Echeverri, Full-genome RNAi profiling of early embryogenesis in Caenorhabditis elegans, Nature 434 (2005) 462–469. [8] C. Jeronimo, D. Forget, A. Bouchard, Q. Li, G. Chua, C. Poitras, C. Therien, D. Bergeron, S. Bourassa, J. Greenblatt, B. Chabot, G.G. Poirier, T.R. Hughes, M.

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