The RNA Pol II sub-complex hsRpb4/7 is required for viability of multiple human cell lines

Abstract

The evolutionarily conserved RNA Polymerase II Rpb4/7 sub-complex has been thoroughly studied in yeast and impacts gene expression at multiple levels including transcription, mRNA processing and decay. In addition Rpb4/7 exerts differential effects on gene expression in yeast and Rpb4 is not obligatory for yeast (S. cerevisiae) survival. Specialised roles for human (hs) Rpb4/7 have not been extensively described and we have probed this question by depleting hsRpb4/7 in established human cell lines using RNA interference. We find that Rpb4/7 protein levels are inter-dependent and accordingly, the functional effects of depleting either protein are co-incident. hsRpb4/7 exhibits gene-specific effects and cells initially remain viable upon hsRpb4/7 depletion. However prolonged hsRpb4/7 depletion is cytotoxic in the range of cell lines tested. Protracted cell death occurs by an unknown mechanism and in some cases is accompanied by a pronounced elongated cell morphology. In conclusion we provide evidence for a gene-specific role of hsRpb4/7 in human cell viability.

Graphical abstract

Keywords

RNA Polymerase II / hsRpb4/7 sub-complex / gene expression / cell viability / cell shape

INTRODUCTION

The Rpb4 and Rpb7 subunits of RNA Polymerase II (Pol II) are of particular interest because, in contrast to other Pol II components, they form a dissociable sub-complex that has specialised gene-specific effects (Choder, 2004; Sampath and Sadhale, 2005). There is significant structural conservation of Rpb4/7 across the animal and plant kingdoms but current understanding of Rpb4/7 function has come mostly from yeast.

S. cerevisiae (sc) Rpb4/7 form a dissociable complex with Pol II (Edwards et al., 1991; Khazak et al., 1998) and scRpb4/7 activates promoter-specific transcription in vitro (Edwards et al., 1991; Jensen et al., 1998). The Rpb4/7 sub-complex also interacts with general transcription factors TFIIB and TFIIF (Bushnell and Kornberg, 2003; Chung et al., 2003), transcriptional activator proteins (Bertolotti et al., 1998; Petermann et al., 1998; Shen et al., 1999; Todorova, 2009) and the Pol II CTD phosphatase Fcp1 (Kimura et al., 2002; Kamenski et al., 2004). Intriguingly non-transcriptional functions of Rpb4/7 have also emerged (Choder, 2004; Sampath and Sadhale, 2005). Rpb4/7 binds to RNA as a heterodimer (Orlicky et al., 2001) and both proteins are intimately involved in coupling of transcription and RNA processing (Mitsuzawa et al., 2003; Runner et al., 2008). Second, Rpb4 (Kimura et al., 2001) and Rpb7 (Khazak et al., 1995; Na et al., 2003) can be found in the cytoplasm with Rpb4 mediating RNA export from the nucleus under stress conditions (Farago et al., 2003) and both Rpb4 and Rpb7 are involved in selective mRNA decay (Lotan et al., 2005; Goler-Baron et al., 2008). In terms of their biological roles, while scRpb4 and scRpb7 may not be identical (scRpb4 is conditional (Woychik and Young, 1989) and scRpb7 is essential (McKune et al., 1993)) both proteins are nonetheless involved in particular stress responses (Choder and Young, 1993; Sheffer et al., 1999; Maillet et al., 1999; Pillai et al., 2001) and are implicated in morphological changes that accompany nutrient starvation (Khazak et al., 1995; Pillai et al., 2003). Consistent with the conditional nature of scRpb4, global gene expression profiling in yeast indicates that a limited number of genes (~2% of the genome) are affected by Rpb4 (Pillai et al., 2003), although other evidence points to a more global effect (Miyao et al., 2001).

In contrast to yeast, human (hs) Rpb4/7 function is not well characterised. Highly variable mRNA levels in several human tissues (Khazak et al., 1995; Schoen et al., 1997) may point to a non-essential and/or specialised function. hsRpb4/7 are also implicated in human cancer. The von Hippel-Lindau (VHL) tumor suppressor protein (Na et al., 2003) directly interacts with and destabilises hsRpb7 via the ubiquitin-proteasome pathway. hsRpb7 also interacts physically (Bertolotti et al., 1998; Petermann et al., 1998; Todorova, 2009) and functionally (Zhou and Lee, 2001) with Ewing's family oncoproteins (Romeo and Dei Tos, 2010; Kovar, 2011) and may play a role in the Ewing's family of tumors. It is therefore of significance to understand the role of hsRpb4/7 in mammalian cell proliferation and survival.

Herein we describe experiments to assess the effect of depleting endogenous hsRpb4/7 in human tissue culture cells. Our results provide evidence for a concerted gene-specific role of hsRpb4 and hsRpb7 in survival of at least some human cells. The cytotoxic effect of hsRpb4/7 depletion precludes scrutiny of any specialised role for hsRpb4/7 in cell lines from Ewing's family tumors.

RESULTS AND DISCUSSION

Identification of effective hsRpb4/7 siRNAs

Multiple synthetic siRNAs targeting different hsRpb4 and hsRpb7 sequences were tested for silencing efficacy using a rapid and sensitive reporter assay employing Rpb4/7-EGFP targets and antibody to EGFP (Chau and Lee, 2007). For hsRpb7 four longer than normal 27mer siRNAs (R7L1–4) were tested because longer siRNAs are more potent (Kim et al., 2005) and thus can be used at concentrations (20 nmol/L) low enough to reduce potential off-target effects (Persengiev et al., 2004). Three of the above siRNAs (R7L1, R7L3 and R7L4) had strong silencing activity at 20 nmol/L (Fig. 1A) and one (R7L4) was very effective at 5 nmol/L (data not shown). Similarly for hsRpb4, three 27mer siRNAs (R4L1–3) were effective at 20 nmol/L (Fig. 1A) and one of these (R4L3) also worked well at 5 nmol/L. R4L3 and R7L4 were used to establish conditions for depletion of endogenous hsRpb4 and hsRpb7. Both R4L3 and R7L4 (at 20 nmol/L) were able to significantly deplete endogenous hsRpb4 and hsRpb7 (Fig. 1B) as detected using affinity purified rabbit polyclonal anti-Rpb7 and anti-Rpb4 antibodies.

hsRpb7/4 expression is coordinated in the nucleus and cytoplasm

Destabilisation of hsRpb7 protein by the tumor suppressor VHL protein (Na et al., 2003) suggests that post-transcriptional/translational events may maintain low hsRpb7 protein levels in mammalian cells. Consistent with this suggestion our attempts to express an exogenous hsRpb7-EGFP fusion (Rpb7e, Fig. 2A) or essentially native hsRpb7 (modified only by a small KT3 epitope tag, Fig. 2B) resulted in poor expression. However co-expression with exogenous hsRpb4e (Fig. 2A) or KT3-tagged hsRpb4 (Fig. 2B) greatly elevated hsRpb7e (or hsRpb7) levels respectively and this effect was specific for hsRpb7 (Fig. 2A). Exogenous hsRpb4 did not noticeably increase endogenous hsRpb7 levels, presumably because exogenous hsRpb4 is expressed in only a fraction of cells and/or endogenous hsRpb7 is sequestered in relatively stable complexes with endogenous hsRpb4.

Similar relative levels of expression of hsRpb4 and hsRpb7 in different human tissues (Khazak et al., 1998) suggests that the endogenous genes/proteins might be coordinately expressed. To examine this possibility we observed protein levels following siRNA-mediated depletion. In the experiment shown, R4L3siRNA decreased Rpb4 and also Rpb7 levels (6.3-fold and 5.4-fold respectively) and similarly R7L4siRNA decreased Rpb7 and also Rpb4 (3.7-fold and 3.6-fold respectively). Thus expression of endogenous hsRpb7 and hsRpb4 proteins is inter-dependent (Fig. 2C). In light of role of Rpb4 in mRNA export in yeast (Farago et al., 2003) we asked whether hsRpb4 increases hsRpb7 RNA levels. Exogenous hsRpb7 RNA transcript levels were monitored by primer extension but no effect of hsRpb4 protein was detected (Fig. 2B). Thus the stimulatory effect of hsRpb4 on hsRpb7 protein levels is exerted after mRNA formation and perhaps most likely via hsRpb7 stabilisation within the Rpb4/7 heterodimer. In this event hsRpb4 binding to hsRpb7 would be required for increasing Rpb7 and the assay described above using exogenous proteins will allow this question to be addressed.

Another significant aspect of Rpb4/7 expression concerns sub-cellular location. While some Rpb4/7 is necessarily nuclear for transcription in both yeast and human cells (Khazak et al., 1998), Rpb4 is also found in the cytoplasm in S. pombe (Kimura et al., 2001), shuttles between the nucleus and the cytoplasm in S. cerevisiae (Selitrennik et al., 2006) and is even predominantly cytoplasmic under some stress conditions (Farago et al., 2003). Previously, immuno-staining of endogenous hsRpb4/7 was not conclusive either due to cross-reacting antigens (for Rpb7) or lack of signal for Rpb4 (Khazak et al., 1998). We have also been unable to clearly detect endogenous hsRpb4/7 in mammalian cells using the antibodies described herein. We therefore employed an exogenous Rpb4-EGFP fusion (Rpb4e) and hsRpb7-red fluorescent protein (Rpb7r) to probe their sub-cellular location (Fig. 3). Consistent with the expression levels for the native proteins (Fig. 2), Rpb7r was only observed in cells expressing Rpb4e, thus confirming the stimulatory effect of hsRpb4 on hsRpb7. In addition to nuclear localisation, some cells showed striking cytoplasmic expression with apparent co-localisation of Rbp4e and Rpb7r and an even distribution throughout the cytoplasm (Fig. 3). A small percentage of cells exhibited highly localised and granular cytoplasmic expression pattern near the nucleus and again with apparent co-localisation of Rbp4 and Rpb7 (Fig. 3). The latter phenomenon may reflect the situation in yeast whereby Rpb4/7 localise to cytoplasmic P bodies (Lotan et al., 2005) that are the site of mRNA decay (Ingelfinger et al., 2002). Overall the subcellular pattern of hsRpb4/7 in human cells appears to share common features with yeast and suggests that both nuclear and cytoplasmic functions have been evolutionarily conserved.

Preliminary Gene Expression Profiling in Rpb7-depleted human cells

Global gene expression profiling (GEP) in yeast (S. cerevisiae) indicates (at least under some conditions) that scRpb4 affects a limited number of genes corresponding to ~2% of the genome (Pillai et al., 2003). To evaluate the global effect of hsRpb4/7 in human cells we performed GEP in Rpb7-depleted HeLa cells using Agilent Human Whole Genome oligo arrays (see materials and methods). Validity of the GEP was verified by scoring of a 77% reduction in hsRpb7RNA in the GEP analysis and determination of a similar reduction (90%) in hsRpb7 protein in depleted versus control cells (Fig. 1A). In Rpb7-depleted HeLa cells GEP resulted in scoring of 429 up-regulated signals or only ~1% of the genome (using a 2-fold threshold) and only 88 up-regulated genes (3-fold threshold). The limited number of genes affected cannot be attributed to partial Rpb7-depletion because expression of eighteen genes was increased between 6-fold and 34-fold. Identification of up-regulated genes is consistent with the role of Rpb4/7 in selective mRNA decay in yeast (Lotan et al., 2005; Goler-Baron et al., 2008). Similarly Rpb7-depletion identified 261 down-regulated signals (2-fold threshold) and only 71 down-regulated genes (2.5-fold threshold). Significant impact on expression of a limited portion of cellular genes further indicates that hsRpb4/7 has a gene-specific rather than general role in human cells.

Because depletion of hsRpb4/7 ultimately leads to cell death (see below) it is not a simple task to identify key primary target genes for hsRpb4/7. Nonetheless mapping differentially expressed genes to the Gene Ontology (GO) database (genes significantly enriched in GO categories, t-test p < 0.05) resulted in selection of several GO categories and some general features may be insightful. Firstly considering the cytotoxic effect of hsRpb7 depletion (see below) it may be significant that several genes implicated in cell cycle arrest/non-apoptotic cell death (CDKN1A, SESN1, HEXIM1) are significantly up-regulated (3–4 folds). Secondly a significant proportion (15%) of genes involved in various biosynthetic/metabolic pathways are altered by hsRpb7-depletion and this may mirror the general defect in expression of metabolic genes in yeast in the absence of Rpb4 (Pillai et al., 2003).

Effect of hsRpb4/7 depletion on human cell viability

Variable mRNA hsRpb4/7 RNA levels in several human tissues (Khazak et al., 1995; Schoen et al., 1997) suggests that hsRpb7 might not be essential for cell survival. As a prerequisite for examining the above question we confirmed expression of hsRpb4 and hsRpb7 proteins in several human cell lines (Jeg3, MST1, Kao, DTC1, GG62) in addition to HeLa (Fig. 4). Both proteins were expressed at reasonably similar levels in nuclear extracts from all cells tested (~3-fold variation, normalised for cell number) and to date we have not identified any cell lines lacking hsRbp4/7. In addition the relative expression of hsRpb4/hsRpb7 is broadly similar comparing different cells (relative signals in Fig. 4 reflect the different antibodies and not the absolute hsRpb4/7 ratio). It thus appears from our preliminary analysis that smaller variations in hsRpb4/7 protein levels in different cell lines may not reflect the more significant differences in mRNA levels observed in human tissues (Khazak et al., 1995; Schoen et al., 1997) or cell lines (K.A.W.L, unpublished results).

Initially we did not observe any cytotoxicity in Hela, Jeg3, DTC1 or GG62 cells following short-term hsRpb4/7 depletion (up to 72hr) using synthetic siRNAs. This prompted us to examine the effect of prolonged depletion via stable introduction of constitutive, selectable short-hairpin (sh) RNAs targeting hsRpb4/7 (Fig. 4). The gene silencing efficacy of different shRNAs against hsRpb4 (R4sh, corresponding to R4L3 siRNA), hsRpb7 (R7sh, corresponding to R7L4 siRNA) and a negative control shRNA (nc) was again established using a transient assay in HeLa cells and scoring depletion of hsRpb7e and hsRpb4e reporter proteins (Fig. 4). For cell viability assays, stable cell transformants were selected by G418 and viable colonies stained with Coomassie Blue (12 days post selection). We first tested two cell lines (Kao and MST1) derived from Soft Tissue Clear Cell Sarcoma (ST-CCS), a member of the Ewing's family of tumors (Zucman et al., 1993; Dim et al., 2007; Wang et al., 2009). Both hsRpb4 and hsRpb7 shRNAs were toxic to Kao (data not shown) and MST1 cells (Fig. 4). However the same result was obtained for two non-ST-CCS cell lines, HeLa and Jeg3 (Fig. 4). The few viable colonies that survive following introduction of shR4/7 presumably arise due to low frequency loss/inactivation of the short-hairpin part of the transgene while retaining neomycin resistance. Together with other observations (see below and Fig. 6) this indicates that the ultimate outcome of prolonged Rpb4/7 depletion is cell death.

To provide rigorous evidence that the cytotoxic effect observed is due to hsRpb7 depletion, we tested a panel of hsRpb7 shRNAs (m2, m6, m7 and m8) with discrete mutations in the 5 region of the siRNA guide strand (Fig. 5). Mutants m6 and m7 retain both Rpb7 silencing and cytotoxicity while mutants m2 and m8 lose both functions (Fig. 5). Thus for several hsRpb7 shRNAs tested there is a correlation between silencing activity and cytotoxicity, indicating that the cytotoxic effect is due to lack of hsRpb4/7. In summary these findings indicate that hsRpb4/7 is required for survival of several human cell lines.

Morphological changes induced by hsRpb4/7 depletion

The eventual outcome of hsRpb4/7 depletion is cell death but our results provide little insight into the mechanism involved. In addition the apparent lack of an acute cytotoxic response to hsRpb4/7-depletion may point to an indirect effect. It is of interest however that in some cells (HeLa) we observed a pronounced elongated cell morphology for both hsRpb4 and hsRpb7-depleted cells that persists for a substantial period (several days) prior to cell death (Fig. 6). Western blot analysis confirmed that hsRpb7 protein levels remain depleted (~6-fold, normalised for cell number) in the above cells prior to cell death (Fig. 6). Because the cells studied normally grow in clusters with close cell-cell contact (even at low density, for example under G418 selection, Fig. 6) it cannot be ruled out that the striking morphology of hsRpb4/7-depleted cells results indirectly from an alteration of cell adhesion properties. The few viable colonies that survive following introduction of shR4/7 look like normal HeLa cells and grow in clusters (i.e. they are completely different compared with the elongated shR4/7 transformants that slowly die). These residual colonies therefore represent background and presumably arise due to low frequency loss/inactivation of the short-hairpin part of the transgene. Thus we suggest that prolonged depletion of Rpb4/7 ultimately leads to cell death.

In relation to the altered morphology of Rpb4/7-depleted HeLa cells it is intriguing that hsRpb7 was originally identified for the ability to enhance pseudohyphal growth in yeast (S. cerevisiae) under nitrogen starvation (Khazak et al., 1995). Such morphological changes are postulated to protect cells by enhancing the probability of finding a nutrient source with pseudohyphal cells being bigger and markedly elongated (Gimeno et al., 1992). S. cerevisiae cells lacking Rpb4 are also more prone to generate pseudohyphae (Pillai et al., 2003) and the fission yeast S. pombe exhibit elongated features and a deficiency in cell separation when Rpb4 expression is low (Sharma et al., 2006). It will be interesting to further characterise the hsRpb4/7-dependent morphological effects that we have described (Fig. 6) and to see if they reflect an evolutionarily conserved response mechanism.

CONCLUSION

We have directly addressed the function of Rpb4/7 in human cells and our results indicate that hsRpb4/7 have gene-specific effects but are also more generally required for human cell survival (at least in cultured cell lines). The latter finding so far precludes examination of a more specialised role of hsRpb4/7 in Ewing's family tumors. Inter-dependence of hsRpb4 and hsRpb7 protein levels and coincident sub-cellular localisation points to the concerted action of hsRpb4 and hsRpb7 via heterodimeric hsRbp4/7. It is likely that stabilisation of hsRpb7 requires interaction with hsRpb4 and the assay described herein, using exogenous hsRpb4/7, will allow the mechanism to be elucidated.

Our results demonstrate that several properties of hsRpb4/7 broadly mirror the well established characteristics of Rpb4/7 in yeast and thus that both nuclear and cytoplasmic Rpb4/7 functions have been evolutionarily conserved. In addition, the pronounced elongated cell morphology observed in hsRpb4 and hsRpb7-depleted cells prior to death may resemble the enhancement of pseudohyphal growth in yeast observed under nitrogen starvation. It will be interest to further characterise the Rpb4/7-dependent morphological effects in mammalian cells to determine whether they reflect an evolutionarily conserved response mechanism.

MATERIALS AND METHODS

Plasmids

Protein expression vectors were derived from pSG424 (Sadowski and Ptashne, 1989) and proteins tagged with the epitope for monoclonal antibody KT3 (MacArthur and Walter, 1984) or from pd2EGFP-N1 expressing destabilised EGFP (Clontech). EGFP-tagged phsR7T and phsR4T express KT3-tagged hsRpb7 and hsRpb4 respectively. pR7E and pR4E express EGFP-tagged hsRpb7/hsRpb4. pN3e expresses the N-terminal 167 residues of the EAD tagged with EGFP. All plasmids expressing shRNAs were constructed by inserting synthetic gel-purified DNA oligonucleotides containing the shRNA sequence between the BamH1 and Hind3 sites of pSilencer 2.1-U6neo (Life Technologies).

Cell lines and cell culture

All cells were maintained in DME and 10% fetal bovine serum. ST-CCS cell lines MST1 and Kao are previously described (Li et al., 2003). For selection of stable transformants DNA was introduced by calcium phosphate/DNA co-precipitation, using 10 μg of pSilencer 2.1-U6neo plasmid as positive control (or test shRNA plasmid) and 10 μg of pGem3 as carrier. At 48 h post-transfection 500 μg/mL G418 was added the culture medium followed by a change of media and fresh drug every two days. Stable colonies were fixed and stained with Coomassie Blue R250.

siRNAs

Synthetic siRNAs contained single strands of 27nt and 29nt producing double stranded siRNAs with a 2-nucleotide (UU) 3' overhang. The sequence for the most effective hsRpb7siRNA (R7L4) is 5'-UGCACAGGGAAGUAUGGCUUUGUAAUUUU-3' (passenger strand) and 5'-AAUUACAAAGCCAUACUUCCCUGUGCA-3' (guide strand). The sequence for the most effective hsRpb4siRNA (R4L3) is 5'-UACACAGCCCGUUUCAGUCGUUUCAAAUU-3' (passenger strand) and 5'-UUUGAAACGACUGAAACGGGCUGUGUA-3' (guide strand). shRNAs expressed from pSilencer 2.1-U6neo were designed to produce in vivo processed siRNAs with guide strands close to or identical with siRNAs R7L4 and R4L3. siRNAs were evaluated in HeLa cells using an EGFP-fusion reporter assay (Chau and Lee, 2007). Briefly cells in 24 well tissue culture plates were co-transfected with plasmids expressing the test protein fused to destabilised-EGFP and pd2EGFP-N1 (300 ng each) and synthetic siRNAs using Lipofectamine 2000 (Life Technologies). At 20 h post-transfection cells were harvested by trypsinisation, washed with PBS and then lysed in buffer containing 20 mmol/L Tris-Cl, pH 7.5; 50 mmol/L NaCl; 1 mmol/L EDTA, pH 8; 1 mmol/L EGTA, pH 7; 1% Triton X-100 and complete protease inhibitor cocktail (Roche). Lysed cells were centrifuged at 13,000 g for 10 min and the cell lysate immediately adjusted to 1× SDS gel loading buffer for Western blot analysis.

Western blotting and primer extensions

Western blotting was performed in PBS containing 3% dried milk. Endogenous hsRpb4 and hsRpb7 were detected using affinity purified rabbit polyclonal antibodies raised against bacterially expressed hsRpb4 and hsRpb7. Exogenous tagged proteins were detected using primary mouse monoclonal antibody KT3 (MacArthur and Walter, 1984) or EGFP antibody (mouse monoclonal JL8, Clontech). Secondary antibodies were anti-mouse or anti-rabbit HRP conjugated (Amersham NA931) and blots were developed using an ECL kit (Amersham NA931). Primer extension of hsRpb7 mRNA was performed as previously described (Lee and Green, 1987) using a 20nt ³²P-labeled primer (5'-CAGGATTTCGTGCTCTAGGG-3') complementary to a sequence near the 5' end (position 127–146) of hsRpb7mRNA.

Gene Expression Profiling (GEP)

GEP analysis was performed by Welgene Biotech Ltd. (Taiwan) employing Agilent Human Whole Genome Oligo 4×44 K arrays. Data were normalized by Rank-invariant LOWESS normalization using Feature Extraction software (Agilent Technologies) with subsequent data analysis using GeneSpring 7.3.1 (Agilent Technologies). For hsRpb7-depleted versus control samples, Rpb7 protein depletion was verified by Western blotting (Fig. 1) and RNA samples were prepared at 72 h post-transfection. For GEP analysis, 429 features (1.05% of total) with “High Confidence Significant UP” (>2-fold, P < 0.01, rProcessed Signal > 100) and 261 features with “High Confidence Significant Down” (<0.5, P < 0.01, gProcessed Signal > 100) were selected.

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