The Conserved Foot Domain of RNA Pol II Associates with Proteins Involved in Transcriptional Initiation and/or Early Elongation [Genetics]
(Genetics Via Acquire Media NewsEdge) ABSTRACT RNA polymerase (pol) II establishes many protein-protein interactions with transcriptional regulators to coordinate different steps of transcription. Although some of these interactions have been well described, little is known about the existence of RNA pol II regions involved in contact with transcriptional regulators. We hypothesize that conserved regions on the surface of RNA pol II contact transcriptional regulators. We identified such an RNA pol II conserved region that includes the majority of the "foot" domain and identified interactions of this region with Mvp1, a protein required for sorting proteins to the vacuole, and Spo14, a phospholipase D. Deletion of MVP1 and SPO14 affects the transcription of their target genes and increases phosphorylation of Ser5 in the carboxyterminal domain (CTD). Genetic, phenotypic, and functional analyses point to a role for these proteins in transcriptional initiation and/ or early elongation, consistent with their genetic interactions with CEG1, a guanylyltransferase subunit of the Saccharomyces cerevisiae capping enzyme.
IN eukaryotes as in archaea, bacteria, chloroplasts, some mitochondria, and nucleocytoplasmic DNA viruses, transcription is ensured by heteromultimeric DNA-dependent RNA polymerases (Thuriaux and Sentenac 1992; Vassylyev et al. 2002; Werner and Weinzierl 2002; Iyer et al. 2006). RNA polymerase II (RNA pol II) produces all mRNAs and many noncoding RNAs. Although it transcribes most of the nuclear genome, it contributes ,10% of the total RNA present in growing cells (Hahn 2004). To transcribe a gene, RNA pol II requires the action of general transcription factors, coregulators, specific transcription activators, and repressors. In fact, the RNA pol II transcription machinery is the most complex of those associated with the three RNA polymerases, with a total of nearly 60 polypeptides (Hahn 2004).
Knowledge of both the architecture making up this complex and the function of its different parts is essential to understand their role in the different transcription steps (Cramer 2006; Zaros et al. 2007; Venters and Pugh 2009). Structural data gathered over the last few years on Saccharomyces cerevisiae RNA pol II have provided a detailed map of the physical interactions between the different subunits, establishing regions that are important for transcription (Cramer et al. 2001; Bushnell et al. 2002; Armache et al. 2003; Meyer et al. 2009). Notably, recent work has contributed to the understanding of how RNA pol II amino acid regions or subunits are involved in the contact with transcriptional regulators such as TFIIS, TFIIB, TFIIE, TFIIF, or Mediator, among others, although the data are sometimes imprecise or controversial (Guglielmi et al. 2004; Chadick and Asturias 2005; Chen et al. 2007; Meyer et al. 2009; Kostrewa et al. 2009).
A major question that remains unexplored is the identification of domains of RNA pol II that could be involved in the interaction with elements of the transcriptional machinery and that could participate in coordinating with them. The identification of new transcriptional regulators, how they assemble in the transcriptional machinery, and their contribution to these processes would be useful.
Here, we describe the existence of a conserved protein domain corresponding to the foot of Rpb1 of S. cerevisiae RNA pol II, located on the surface of the complex. We have identified interactions of this region with Mvp1 and Spo14 and demonstrate that a fraction of these proteins localizes in the nucleus. ChIP-chip analysis suggests that both Mvp1 and Spo14 associate with RNA pol II genes, but not with RNA pol I or III genes. Deletion of MVP1 and SP014 affects the expression of some of their target genes, as well as genes regulated by Mot1 and/or NC2 and increases phosphorylation of Ser5 in the carboxy-terminal domain (CTD), consistent with the genetic interactions between Dmvp1 or Dspo14 and the Drtr1 mutation. Furthermore, these data together with phenotypic and functional analysis point to a role for these proteins in transcription initiation and/or early elongation, in accordance with the genetic interactions with CEG1. In addition, our data clearly agree with data from Suh and coworkers that have also defined the foot of the RNA pol II as a domain conserved among RNA pol IIs from different species and that contact the RNA capping enzyme (CE) in S. cerevisiae (Suh et al. 2010).
Materials and Methods
Yeast strains, plasmids, genetic manipulations, media, and genetic analysis
Common yeast media, growth conditions, and genetic techniques were used as described elsewhere (Garcia-Lopez et al. 2010).
Strains and plasmids are listed in Tables 1 and 2, respectively. MAY322 strain (Biomedal), expressing the C-LYTAG domain from the NHP6A gene promoter, was obtained from strain BY4742 by replacing the NHP6A ORF with the C-LytA ORF, through chromosomal integration of a PCR product from plasmid pUC19-lytAstop-cyc1term-His3MX6.
Two-hybrid screening and identification of interacting proteins
The FRYL genomic library (Fromont-Racine et al. 1997) contained randomly sheared genomic DNA fragments of 700-bp mean size in a modified pACT2 vector. Two-hybrid analyses were as described (Flores et al. 1999). The prey DNA were amplified by PCR and sequenced with 242 and 244 primers (Supporting Information, Table S2). The identity of the insert was determined by using the Saccharomyces Genome Database Blast service.
Proteins were tagged with a C-LYTAG tag (Biomedal) as described in Longtine et al. (1998) amplified from the pUC19-LytA-Kan plasmid (gift from S. Chávez) by PCR, with primers MVP1lyt-501/301 and SPO14lyt-501/301. Positive colonies were analyzed by PCR with the Mvp1-501/301 and Spo14-501/301 primers (Table S2).
Immunoprecipitations (IPs) were carried out as described (Soutourina et al. 2006) with 100 ml of protein extracts (1500 mg) prepared from cells growing exponentially (A600 0.6-0.8) in yeast extract-peptone-dextrose (YPD) medium. An anti-C-LYTAG antibody (50 ml at 1 mg/ml) (Hernandez-Torres et al. 2008) was used. The affinity-purified proteins were released from the beads by boiling for 10 min. Eluted proteins were analyzed by Western blotting with anti-C-LYTAG and anti-Rpb1 (gift from P. Thuriaux) antibodies.
Cells were grown at 30 in SD medium (A600 0.8-1.0), fixed with 37% w/v formaldehyde at room temperature for 1 hr with slow shaking, and then centrifuged and washed twice with PBST (PBS 1 with 0.05% Tween-20). Cell wall was digested with 50 mg/ml zymolyase in PBST (USBiological) by incubation for 30 min at 37 without shaking. The spheroplasts were washed twice with PBST and then resuspended in the same solution. Cell suspension was added to an AAS (3-aminopropyltriethoxysilane; Sigma) slide, incubated at room temperature for 15 min and washed twice with PBST. A total of 50 ml of 1:50 dilution of the anti-C-LYTAG primary antibody in PBST-BSA (5 mg/ml BSA) were added and incubated overnight at 4. The slides were then washed twice with PBST-BSA and incubated for 2 hr, in the dark, at room temperature, with 50 ml of 1:300 dilution of secondary antibody (anti-rabbit IgG conjugated with Cy3; The Jackson Laboratory). The slides were washed twice with PBST-BSA and finally covered with a Vectashield (Vectorlabs) mounting solution. The fluorescence intensity was scored with a fluorescence microscope (Olympus BX51).
For ChIP-chip experiments we followed the protocol described in Jimeno-Gonzalez et al. (2006) but using antibodies against C-LYTAG epitope. We included no-antibody samples (NA) as the negative controls of the immunoprecipitation process. Two independent biological replicates were made. Specificity for the candidate genes was reconfirmed by Q-PCR. Each PCR reaction was performed three times (Table S2 for oligonucleotides).
For Rpb1 (non-P-CTD), Ser5P, and Ser2P IPs, 8WG16 (Covance), CTD4H8 (Millipore), and ab5095 (Abcam) antibodies were used and chromatin immunoprecipitations were performed as previously described (Garcia et al. 2010). Genes were analyzed by quantitative real-time PCR in duplicate with at least three independent biological replicates. Values found for the immunoprecipitated PCR products were compared to those of the total input, and the ratio of values from each PCR product of transcribed genes to that of a nontranscribed region of CVII was calculated. The oligonucleotides used are listed in Table S2.
DNA amplification and array hybridization
Ligation-mediated PCR (LM-PCR) (Ren et al. 2000) was applied for DNA amplification and the PCR product labeled with 33P-dCTP as described in Pelechano et al. (2009) with oligonucleotides Linker LE59 oJW102 and oJW103. Radioactive samples were hybridized onto macroarrays on which PCR products representing full-length ORFs for 6049 genes of S. cerevisiae were spotted (Ren et al. 2000) (Servei Central de Suport a la Investigació Experimental, Universitat de València, Spain).
Image analysis and data normalization
Image analysis and data normalization were undertaken as described (Pelechano et al. 2009). Images were quantified using the ArrayVision software 7.0 (Imaging Research). The signal intensity for each spot was the background subtracted ARM Density (artifact-removed median). Only enrichment values 1.35 times above the background were considered valid. Reproducibility of the replicates was checked using the Array- Stat software (Imaging Research). Normalization between conditions was performed by the global median method and the ratio between IP and whole cell extract (WCE) in each experiment was taken as the binding ratio. The functional analyses of the IP data were made using the Fatiscan application from Babelomics (Al-Shahrour et al. 2007). The genomic data are stored in Valencia Yeast (VYdBase; http://vydbase.uv.es/) and GEO databases (GSE16905).
Extraction of mRNA and reverse transcription
Total RNA from yeast cells and reverse transcribed RNAs were prepared as previously described (Garcia-Lopez et al. 2010).
Quantitative real-time PCR
To analyze gene expression, cDNA corresponding to 0.5 ng of total RNA was used. Each PCR reaction was performed at least three times, with three independent samples. The 18S rRNA and the ACT1 genes were used as the normalizers. The amplified PCR products were verified by agarose gel electrophoresis.
Sequence alignments were based on a saturating homology search with the standard default Psi-Blast and Multalin (Corpet 1988; Schaffer et al. 2001); see http://blast.ncbi.nlm. nig.gov/ and http://bioinfo.genotoul.fr/multalin/multalin.html. In some cases, they were improved by visual inspection, based on the following amino acid conservations: AG, ST, CS, DN, DE, EQ, MILV, KR, and FWY.
Statistical data analysis
Samples were compared by the Student's t-test using the Statgraphics Plus program.
Identification of conserved domains of the RNA polymerase II
To identify conserved domains of the RNA pol II potentially involved in the interaction with transcriptional regulators, we searched for regions located on the surface of the structure of the complex that were also conserved among different species, but with poor or no conservation in their paralogs in RNA polymerases I (Rpa190) and III (Rpc160) or in their homologs in archaea and bacteria.
Using PSI-Blast and Multalin (Corpet 1988; Schaffer et al. 2001), we carried out an amino acid sequence alignment between the largest subunit of the RNA pol II from S. cerevisiae, Rpb1, its orthologs in different eukaryotic species, its paralogs Rpa190 and Rpc160, and its homologs in archaea and bacteria (see García-López and Navarro 2011).
We identified a region of 163 amino acids (residues 881- 1044 of S. cerevisiae Rpb1) conserved in all Rpb1 sequences (Figure 1A). This region, designated as the "conserved domain of the foot," corresponds to the helix a27-a34, which includes most of the domain previously called the foot (Cramer et al. 2001; García-López and Navarro 2011) (Figure 1B). Consistently, the Rpb1 foot is poorly conserved in the structure of the RNA polymerase III from S. cerevisiae (Fernandez-Tornero et al. 2007).
Interactions with the conserved domain of the foot of the RNA pol II
The conserved domain of the foot was fused to the Gal4pBD in the pGBT9 vector and introduced into the tester strain Y190, which has two reporter genes for two-hybrid interaction, GAL1::lacZ and GAL(UAS)::HIS3. As a control, we also fused the same region to the Gal4pAD in the pACT2 vector and introduced it in the tester strain Y187.
The fusion protein did not confer resistance to 50 mM 3AT, indicating that it did not operate as transcriptional activators of Pol II, and thus could be used in the screen. In addition, no b-galactosidase activity was observed with any of the negative controls used in the experiment (Figure 2A).
We tested 2.5 107 transformants (at 50 mM 3AT) with a similar efficiency (37%) to other previous screens performed with the same library (Flores et al. 1999). Five 3ATR bGal+ clones were obtained (Figure 2). One of the interacting preys was a domain spanning the last 79 amino acids of Mvp1 (413-511) and the second, 258 amino acids in the C terminus of Spo14 (residues 1304-1562; the protein is 1683 amino acids long).
Mvp1, a protein required for sorting proteins to the vacuole (Ekena and Stevens 1995), physically interacts with three nuclear proteins, Std1, Yra2, and Srb7 (Schmidt et al. 1999; Hazbun et al. 2003; Vollert and Uetz 2004; Titz et al. 2006). Similarly, Spo14, a phospholipase D involved in Sec14pindependent secretion and required for meiosis and spore formation (Rudge et al. 2002; Nakanishi et al. 2006), physically interacts with Dcp2, (Fromont-Racine et al. 1997) and genetically interacts with the transcription factor Ste12 (Hairfield et al. 2001). The interactions of Mvp1 and Spo14 with the conserved domain of the footmight reveal connections between transcription and other aspects of the nuclear metabolism.
Mvp1 and Spo14 bind RNA pol II in vivo
Mvp1 and Spo14 were tagged at their C terminus by inserting a sequence encoding the C-LYTAG domain of the lytA gene from Streptococcus pneumoniae (17 kDa). The addition of C-LYTAG tag to these nonessential proteins did not affect the growth of these strains.
Mvp1-C-LYTAG and Spo14-C-LYTAG were immunoprecipitated with anti-C-LYTAG antibody. In both cases, an Rpb1 reacting band was also revealed (Figure 3, right). No such band was observed when the IPs were performed in control strain MAY322 (Figure 3, right), indicating that Rpb1 does not interact with the C-LYTAG module and that no anti- Rpb1 reacting material was immunoprecipitated nonspecifically by anti-C-LYTAG antibody or adsorbed nonspecifically to the IgG magnetic beads. Similar results were found when C-LYTAG proteins were purified from Mvp1-C-LYTAG tagged and MAY322 strains by using a DEAE-cellulose matrix (data not shown). These observations suggest that interactions between RNA pol II and Mvp1 or Spo14 are specific.
Mvp1 and Spo14 localize in the nucleus of S. cerevisiae
We performed immunocytochemistry experiments with anti-C-LYTAG antibodies, using the Mvp1-C-LYTAG and Spo14-C-LYTAG version of the proteins. As shown in Figure 4, we detected mostly a cytosolic signal for both proteins. However, we also observed a nuclear staining. On the contrary, a control for C-LYTAG localization using the MAY322 strain showed only cytosolic signal (Figure 4), indicating that the nuclear localization of Mvp1 and Spo14 is not artifactual.
These data together indicate that fractions of Mvp1 and Spo14 are localized inside the S. cerevisiae nucleus, probably according to their physical interaction with RNA pol II.
Mvp1 and Spo14 associate only with RNA pol II genes and regulate expression of their targets
ChIP-chip experiments were performed with Mvp1-CLYTAG and Spo14-C-LYTAG. The signals were ordered by intensity after normalizing the results with total DNA. The global IP results as well as the genes with higher IP enrichment are shown in Table S1. The control probes for RNA pol I and RNA pol III genes were among the lowest intense spots, meaning that both proteins do not bind those kinds of genes. Given the difficulty of establishing a threshold separating bound and unbound genes, we looked for enriched Gene Ontology categories using a scanning algorithm (Fatiscan; Al- Shahrour et al. 2007). Spo14 was enriched on ribosomal protein genes (Figure 5A), whereas Mvp1 was not associated with any particular GO category. By performing Q-PCR ChIP assays [using probes encompassing the 59 regions of the genes (surrounding ATG) or inside the genes], we confirmed independently the association of Mvp1 or Spo14 with those genes most strongly enriched in the ChIP-chip experiments (MBF1 and RPS15 for Mvp1 and DAL3 and LSM8 for Spo14) (Figure 5B).
We also compared the binding level of Mvp1 and Spo14 with the dataset of nascent transcription rates (TRs) calculated by genomic run-on (Pelechano et al. 2010). As can be seen in Figure 6A, there is a significant positive relationship between gene binding and TR for both proteins.
As the association of Mvp1 and Spo14 to specific genes suggested that they may act as transcription factors, we next investigated whether these proteins could regulate the expression of their target genes. The deletion of MVP1 and SPO14 clearly altered the expression of MBF1 and also of RPS15, LSM8, and PMA1, although to a lesser extent (Figure 6B). However, this is not a general effect on gene expression, as PYK1 or ACT1 expression (used as internal control; data not shown) remains similar to those observed in a wild-type strain.
Phenotypic and genetic analyses indicate that Mvp1 and Spo14 have a role in transcription
Because Mvp1 and Spo14 were identified as interactors with RNA pol II, we considered the possibility that these proteins are functionally linked to each other. Figure 7, A-C show the most relevant phenotypes for the mutants analyzed and Figure 7D the whole genetic analysis. Deletion of MVP1 but not of SPO14 shows a synthetic growth defect when combined with the rpo21-4 mutation in the conserved domain of the foot (Figure 7, A and D). This genetic interaction is specific to this rpb1 mutant, since no differences in growth were detected when Dmvp1 was combined with another rpb1 or rpb2 mutation.
In an attempt to clarify the role of Mvp1 and Spo14 in transcription, we explored whether these proteins participated in transcription elongation. As opposed to other S. cerevisiae strains that are defective for transcription elongation and often sensitive to 6-azauracil (6AU) andmycophenolic acid (MPA) (Shaw et al. 2001; Garcia-Lopez et al. 2010), Dmvp1 and Dspo14 mutants were not sensitive to these drugs (see Figure 7B). In addition, the deletion of MVP1 and SPO14 does not affect mRNA biogenesis efficiency measured by GLAM, a method previously used as an indirect estimation of RNA pol II elongation (Morillo-Huesca et al. 2006). Although these negative results are not sufficiently strong to discard a relationship with transcription elongation, our data could suggest that these proteins are not involved in this process. However, the deletion of these genes corrected the growth and the sensitivity to 6AU and MPA of a Ddst1 mutant (for TFIIS elongation factor) affected in transcription elongation and initiation (Kim et al. 2007; Guglielmi et al. 2007; Ghavi-Helm et al. 2008) (Figure 7, A, B, and D).
Similarly, the deletion of SPO14 suppressed the slowgrowth phenotype and the MPA sensitivity of a mutant deleted for RPB4, a nonessential specific subunit of the RNA pol II participating in transcription initiation, elongation, or mRNA export (Choder 2004; Goler-Baron et al. 2008) (Figure 7, A, B, and D). RPB4 and DST1 genetically interact (Wery et al. 2004), as do RBP9 and DST1, another RNA pol II nonessential subunit regulating transcription initiation and elongation. However, deletions of MVP1 or SPO14 did not alter the growth of cells lacking RPB9.
We also tested for conditional synthetic interactions between Dmvp1 and Dspo14 and mutations of the transcriptional initiation machinery. As shown in Figure 7, C and D, MVP1 and SPO14 genetically interact with BUR6, a component of the negative cofactor 2 (NC2) and MOT1, two conserved regulators of TATA-binding protein (TBP) function that cooperate to regulate gene expression on a global scale (Geisberg et al. 2001; Dasgupta et al. 2005; Masson et al. 2008; Van Werven et al. 2008). These data suggest that Mvp1 and Spo14 could be involved in any step of the transcriptional initiation or early elongation.
Mvp1 and Spo14 regulate transcription from HSP12 and HSP26 promoters
Considering the genetic interactions between MVP1, SPO14, and elements of the transcription initiation machinery, we examined the transcriptional activity of the HSP12 and HSP26 promoters, that are regulated by NC2 and/or Mot1, respectively (Creton et al. 2002; Dasgupta et al. 2005; Peiro- Chova and Estruch 2007; Masson et al. 2008). Deletion of MVP1 and SPO14 decreased HSP12 and HSP26 mRNA accumulation by 50% at 30 (Figure 8A) and significantly altered the induction levels of HSP12 and HSP26 promoters when cells were shifted to 37 for 30 min (Figure 8B). This effect is not a general consequence of the deletion of MVP1 and SPO14 on RNA pol II activity. Moreover, inducible gene expression of GAL1 (not shown), or the constitutive expression of PYK1 and ACT1 genes (see above) were not altered. It is possible that GAL1 is not a target for Mvp1 and Spo14, in concordance with the fact that we did not observed association of these proteins with GAL1 promoter or coding region (data not shown).
Abnormal CTD Ser5P phosphorylation caused by Mvp1 and Spo14 inactivation
Our data account for a connection between Mvp1 and Spo14 and the transcription initiation or early elongation. The Rpb1 CTD is predominantly phosphorylated on serine 5 (Ser5) during promoter scape and early elongation (Komarnitsky et al. 2000; Gu et al. 2010; Mayer et al. 2010). Then we tested whether the deletion of MVP1 and SPO14 would have an effect on Ser5 CTD phosphorylation. We performed ChIP on wild-type, Dmvp1, and Dspo14 cells using antibodies, which recognize unphosphorylated CTD, Ser5 phosphorylated, and Ser2 phosphorylated CTD. As shown in Figure 9A, Ser5P crosslinking increased at promoters of MBF1, RPS15, PMA1, HSP26, and HSP12 genes in both mutants, while no differences with respect to the wild-type strain was noted for the PYK1 gene, which was not affected by the deletion of MVP1 or SPO14. In addition, this phenomenon is specific for CTD Ser5P in the promoter region, since no significant differences for CTD Ser5P were generally observed at the 39 region, nor for CTD Ser2P (Figure 9, B and C). These results also clearly agree with the genetic interactions found between Dmvp1 or Dspo14 strains and the Drtr1 mutant deleted for the gene coding for the recently described Ser5 CTD phosphatase Rtr1 (Mosley et al. 2009) (Figures 7D and 9D).
Ser5 phosphorylation occurs first in CTD in coordinated recruitment of the guanylyl-transferase subunit of the S. cerevisiae mRNA-capping enzyme (Ceg1) (Gu et al. 2010). As expected, MVP1 and SPO14 genetically interact with CEG1 (Figure 9D). Furthermore MVP1 deletion did not alter the growth of truncated mutants of the CTD (see Figure 7D), although these mutations are lethal when combined with a ceg1 mutant (Cho et al. 1997).
In this work, we looked for conserved regions on the surface of the RNA pol II of S. cerevisiae, hypothesizing that they contact transcriptional regulators. We identified a domain at the foot of RNA pol II and demonstrated its interaction with Mvp1 and Spo14. Our study provides physical, genetic, and functional evidence that Mvp1 and Spo14 are associated with the transcriptional machinery and participate in transcription initiation and/or early elongation.
Mvp1 and Spo14 physically interact with the conserved domain of the foot of RNA pol II and localize in the nucleus
We identified a region of 163 amino acids (residues 881- 1044 of S. cerevisiae Rpb1) with a significant conservation (28%) in all Rpb1 subunits, from yeast to human, but with low or no conservation in Rpb1 paralogs and in their homologs in archaea and bacteria. This region, designated as the conserved domain of the foot, corresponded to the majority of the RNA pol II "foot," which in cooperation with the "lower jaw," the "assembly" domain, and the "cleft" regions, constitute the "shelf" module of the RNA pol II that might contribute to the rotation of the DNA as it advances toward the active center (Cramer et al. 2001; Zaros et al. 2007). Consistently, the foot is poorly conserved in the structure of the RNA polymerase III from S. cerevisiae (Fernandez-Tornero et al. 2007). In accordance, Suh and coworkers have also defined the foot of the RNA pol II as an RNA pol II conserved domain (Suh et al. 2010).
We identified interactions between the conserved domain of the foot and Mvp1 and Spo14. It is important to note that these interactions have never before been found in other genomic screens. Interestingly, Suh and coworkers have recently demonstrated that the foot of the RNA pol II contacts the RNA CE in S. cerevisiae (Suh et al. 2010).
Mvp1, a protein required for vacuolar protein sorting in yeast (Ekena and Stevens 1995) and Spo14, a phospholipase D (Rudge et al. 2002; Nakanishi et al. 2006), have never been reported before as proteins participating in transcription. However, physical interactions have been observed between Mvp1 and three nuclear proteins, Std1, Yra2, and Srb7 (Schmidt et al. 1999; Hazbun et al. 2003; Vollert and Uetz 2004; Titz et al. 2006). Similarly, Spo14 physically interacts with Dcp2 (Fromont-Racine et al. 1997) and genetically with the transcription factor Ste12 (Hairfield et al. 2001).
Curiously, Mvp1 and Spo14 are two of 15 proteins containing phox homology (PX) domain in yeast, which is involved in protein-protein interactions (Sato et al. 2001; Vollert and Uetz 2004). However, the regions of Mvp1 and Spo14 in contact with Rpb1 do not contain the PX domains. In addition, no homology between the regions of interaction was detected, thus ruling out the possibility of unspecific interactions due to a similar protein domain in both Mvp1 and Spo14 peptides. In addition, in no case were interactions between these 15 PX domain proteins and Rpb1 observed (Vollert and Uetz 2004).
In agreement with the interactions of Mvp1 and Spo14 with the RNA pol II, we observed that a fraction of Mvp1 and Spo14 localized in the nucleus of S. cerevisiae, although previous data have indicated a cytoplasmic localization of both proteins (Ekena and Stevens 1995; Rudge et al. 1998, 2001). Moreover, the GFP-tagged yeast strain collection show that these proteins give cytoplasmic signals (Huh et al. 2003). Hence, it is possible that the tag we used (CLytag from S. pneumoniae) gave a better and clearer signal, allowing us to detect Mvp1 and Spo14 proteins in the nucleus. This nuclear localization seems not to be artifactual, since a control for C-LYTAG localization using the MAY322 strain in which the C-LYTAG tag is expressed under the control of the NHP6A promoter, shows only cytosolic signal. In addition, the C-LYTAG domain does not contain any NLS or NES motifs, as revealed by using different prediction servers. These data also agree with the interactions of Mvp1 and Spo14 with proteins of the transcriptional machinery (see above). Other proteins that participate in transcription have been also shown to shuttle between the cytoplasm and nucleus in yeast or mammalian cells, such as Rpb4, RMP, or Iwr1, among others (Delgermaa et al. 2004; Peiro-Chova and Estruch 2009; Harel-Sharvit et al. 2010) or even the two Mvp1 interactors Yra2 and Std1 (Vollert and Uetz 2004).
Mvp1 and Spo14 associate with RNA pol II genes and affect gene expression of their target genes
Mvp1 and Spo14 associate with the DNA in vivo, as shown in our ChIp-chip analysis, probably through their interactions with RNA pol II. This occupancy is observed only in RNA pol II genes and, while Spo14 is selectively bound to ribosomal protein genes, Mvp1 does not bind any particular GO category. The fact that only a small fraction of these proteins is detected in the nucleus and the possibility that Mvp1 and Spo14 could associate only transiently with Rpb1 in vivo could account for the low levels of these two proteins detected by ChIP, as observed for other proteins known to play important roles in transcription, such as Mediator (Andrau et al. 2006; Fan et al. 2006; Zhu et al. 2006; Soutourina et al. 2011). According to these data, the deletion of MVP1 and SPO14 affect mRNA accumulation of some of their target genes, although this is not a general feature, so that the expression of other genes such as PYK1 or ACT1 is not altered.
Phenotypic and genetic analyses suggest a role for Mvp1 and Spo14 in any step of transcription initiation and/or early elongation
The genetic interactions with BUR6, a component of the negative cofactor 2 (NC2) and MOT1, two conserved regulators of TBP function that cooperate to regulate gene expression on a global scale (Van Werven et al. 2008), suggest a link between these two proteins and the transcription initiation machinery. These data also agree with the fact that deletion of MVP1 and SPO14 affects the transcription of HSP12 and HSP26 genes regulated by NC2 and Mot1 (Creton et al. 2002; Dasgupta et al. 2005; Peiro-Chova and Estruch 2007; Masson et al. 2008).
In addition, both MVP1 and SPO14 genetically interact with DST1 (TFIIS) and RPB4, two transcriptional elements that also interact genetically (Wery et al. 2004) and that have been shown to participate in transcription initiation (Armache et al. 2003; Choder 2004; Guglielmi et al. 2007; Kim et al. 2007; Goler-Baron et al. 2008; Ghavi-Helm et al. 2008; Garcia-Lopez et al. 2010). All these data together and the fact that MVP1 and SPO14 disruptions do not affect transcription elongation, nor CTD Ser2P, which is associated mainly with elongating RNAPII (Kim et al. 2009; Garcia et al. 2010), argue for a connection between Mvp1 and Spo14 with the transcription initiation machinery, although we cannot rule out a role in early elongation.
Both possibilities are also supported by our data showing that deletion of MVP1 and SPO14 specifically increase CTD Ser5P phosphorylation, and by the genetic interactions between MVP1, SPO14, and RTR1, the gene coding for the recently reported CTD Ser5P phosphatase Rtr1 required for the Ser5-to-Ser2P transition (Mosley et al. 2009). In fact, RNA pol II with CTD Ser5P is generally found in the 59 region of genes at transcription initiation (Kim et al. 2009), but also during promoter escape and early elongation (Mayer et al. 2010).
CTD Ser5P phosphorylation achieved by Kin28 mediates cotranscriptional recruitment of Ceg1 (Gu et al. 2010). In accordance, MVP1 and, SPO14 genetically interact with CEG1. Curiously, the foot of the RNA pol II contacts the RNA CE in S. cerevisiae (Suh et al. 2010). In addition, the mRNA cap-binding complex stimulates the formation of PIC via its interaction with Mot1 in vivo (Lahudkar et al. 2010). It bears noting that the human homolog of Rtr1, RPAP2, has been found as part of the RNA pol II assembly intermediates (Boulon et al. 2010). Consequently, we cannot rule out the possibility that the genetic interactions between MVP1, SPO14, and RTR1 could be associated with a defect in RNA pol II assembly, although this seems unlikely since the deletion of MVP1 and SPO14 do not affect the amount of Rpb1 (data not shown).
In conclusion, all together these data constitute the first experimental evidence pointing to a role of Mvp1 and Spo14 in transcription initiation and/or early elongation. In any case, we cannot rule out that Mvp1 and Spo14 could modulate or allow the access of the Ser5 kinase or Ser5 phosphatases to the transcription complex, as is the case for Abd1 (a capping enzyme in budding yeast) for which inactivation causes a defect in promoter clearance and/or early elongation, which correlates with failure to dephosphorylate Ser5 residues normally (Schroeder et al. 2004). In addition, we cannot dismiss the possibility that the genetic interaction observed between MVP1, SPO14, and DST1 could be related to the fact that TFIIS binds adjacent to the foot domain in the RNA Pol II- TFIIS complex crystal structure (Kettenberger et al. 2003). Finally, we cannot rule out the possibility that Mvp1 and/or Spo14 could participate also in other steps of transcription.
We thank the laboratory of DNA chips of the Servei Central de Suport a la Investigació Experimental de la Universitat de València for making the DNA macroarrays. We thank S. Buratowski, A. L. Harkins, M. C. Schmidt, P. Thuriaux, R. Kellogg, F. Estruch, and R. Young for their kind gifts of strains and plasmids. This work was supported by grants from the Spanish Ministry of Education and Science, Ministry of Sciencie and Innovation, and Fondo Europeo de Desarrollo Regional (FEDER) (BFU2007-67575-C03-03/BMC, BFU2010-21975- C03-02 Spain) and Junta de Andalucía (BIO258, P08-CVI- 03508) (to F.N.), BFU2007-67575-C03-01/BMC (to J.E.P-O.), and BFU2009-07179 (to O.C.). A.I.G.-G. was a recipient of predoctoral fellowships from Universidad de Jaén and currently from Ministry of Education and Culture. A.G. was supported by a fellowship from the Junta de Castilla y León.
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Communicating editor: K. M. Arndt
M. Carmen García-López,* Vicent Pelechano,[dagger],§ M. Carmen Mirón-García,* Ana I. Garrido-Godino,* Alicia García,** Olga Calvo,** Michel Werner,[double dagger] José E. Pérez-Ortín,[dagger] and Francisco Navarro*,1
*Departamento de Biología Experimental, Facultad de Ciencias Experimentales, Universidad de Jaén, 23071 Jaén, Spain,
[dagger]Departamento de Bioquímica y Biología Molecular, Universitat de València, E46100 Burjassot, Spain, [double dagger]Commissariat a la Énergie Atomique, iBiTec-S, Service de Biologie Intégrative et Génétique Moléculaire, F-91191 Gif-sur-Yvette Cedex, France, §Department of Genome Biology, EMBL, D-69117 Heidelberg, Germany, and **Instituto de Biología Funcional y Genómica, Consejo Superior de Investigaciones Cientfícas/Universidad de Salamanca, Campus Miguel de Unamuno, Salamanca 37007, Spain
Copyright © 2011 by the Genetics Society of America
Manuscript received July 25, 2011; accepted for publication September 19, 2011
Supporting information is available online at http://www.genetics.org/content/
1Corresponding author: Departamento de Biología Experimental, Facultad de Ciencias
Experimentales, Universidad de Jaén, Paraje de las Lagunillas, s/n, 23071 Jaén, Spain.
(c) 2011 Genetics Society of America
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