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. 2008 Jul 31;27(17):2317–2327. doi: 10.1038/emboj.2008.151

The plant-specific TFIIB-related protein, pBrp, is a general transcription factor for RNA polymerase I

Sousuke Imamura 1, Mitsumasa Hanaoka 1, Kan Tanaka 1,2,a
PMCID: PMC2529366  PMID: 18668124

Abstract

TFIIB and BRF are general transcription factors (GTFs) for eukaryotic RNA polymerases II and III, respectively, and have important functions in transcriptional initiation. In this study, the third type of TFIIB-related protein, pBrp, found in plant lineages was characterized in the red alga Cyanidioschyzon merolae. Chromatin immunoprecipitation analysis revealed that CmpBrp specifically occupied the rDNA promoter region in vivo, and the occupancy was proportional to de novo 18S rRNA synthesis. Consistently, CmpBrp and CmTBP cooperatively bound the rDNA promoter region in vitro, and the binding site was identified at a proximal downstream region of the transcription start point. α-Amanitin-resistant transcription from the rDNA promoter in crude cell lysate was severely inhibited by the CmpBrp antibody and was also inhibited when DNA template with a mutated CmpBrp–CmTBP binding site was used. CmpBrp was shown to co-immunoprecipitate and co-localize with the RNA polymerase I subunit, CmRPA190, in the cell. Thus, together with comparative studies of Arabidopsis pBrp, we concluded that pBrp is a GTF for RNA polymerase I in plant cells.

Keywords: pBrp, plant, rDNA, RNA polymerase I, TFIIB

Introduction

The general class II transcription machinery is composed of RNA polymerase II (Pol II) and six general transcription factors (GTFs): TFIIA, TFIIB, TFIID, TFIIE, TFIIF and TFIIH (Hahn, 2004). As the first step in preinitiation complex (PIC) assembly, TFIID, which includes the TATA-binding protein (TBP) and TBP-associated factors (TAFs), binds to the core promoter, and the DNA–protein complex is subsequently stabilized by TFIIB. TFIIB is a monomeric protein containing two imperfect direct repeats in the carboxy-terminal region (Deng and Roberts, 2007). These two repeats can directly form a sequence-specific interaction with a core promoter element known as BRE (TFIIB recognition element) (Lagrange et al, 1998; Deng and Roberts, 2005, 2007). The amino-terminal region of TFIIB contains a zinc-chelating motif, which is conserved in all TFIIBs identified to date, and is indispensable for recruitment of the Pol II–TFIIF complex to form PIC (Tubon et al, 2004). The carboxy-terminal proximal to the zinc-ribbon motif is the most conserved region of TFIIB, and is termed as the B-finger. The X-ray crystallography of a yeast Pol II–TFIIB complex revealed that the B-finger domain of TFIIB is inserted into the presumed RNA exit channel of Pol II (Bushnell et al, 2004). It was also revealed that the B-finger has an important function after PIC formation and transcription start site selection (Bushnell et al, 2004; Deng and Roberts, 2007). In addition to TFIIB, the eukaryotic genome encodes another TFIIB-related protein, BRF; this is a component of the TFIIIB complex, which is the core RNA polymerase III (Pol III) initiation factor by virtue of its role in recruiting Pol III to the transcriptional start site, and is essential to form the transcription-ready open promoter complex (Kassavetis and Geiduschek, 2006). Therefore, TFIIB and BRF has an important function in Pol II- and Pol III-dependent transcription during the initiation processes.

Recently, Lagrange et al (2003) reported that higher plant genomes encode a plant-specific TFIIB-related protein, pBrp, as well as TFIIB and BRF. The structure of pBrp contains two TFIIB features: an amino-terminal zinc-ribbon motif and carboxy-terminal imperfect direct repeats. Interestingly, in contrast to known GTFs, the majority of Arabidopsis thaliana pBrp (AtpBrp) and Spinacea oleracea pBrp proteins were bound to the cytoplasmic face of the plastid envelope membrane in leaves. AtpBrp was detected in the nucleus either when the Arabidopsis cell was treated with the proteasome inhibitor, MG132, or in a fus6 mutant, in which the COP9 signalosome is deficient. Thus, it was proposed that the AtpBrp protein harbours a proteolytic signal that can target it for rapid turnover by the proteasome-mediated protein degradation pathway in the nucleus. It was also shown that AtpBrp and AtTBP2 can cooperatively form a ternary complex in vitro with the adenovirus type 2 major late (Ad2ML) promoter, which harbours consensus BRE- and TBP-binding motifs. Therefore, their data suggested the possible involvement of AtpBrp in intracellular signalling between plastids and the nucleus. However, the relevant function in plant cells of the third type of TFIIB-related protein, pBrp, remained unknown.

Cyanidioschyzon merolae is a thermo-acidophilic unicellular red alga isolated from an Italian volcanic hot spring (Kuroiwa, 1998). Recently, a genome project team, including ourselves, determined the 100% complete DNA sequence of this organism including the nuclear and two organelle genomes (Ohta et al, 1998, 2003; Matsuzaki et al, 2004; Nozaki et al, 2007). Because of its extremely simple cell structure and the minimally redundant small genome, this alga is considered to be a suitable model to study the origin and evolution of photosynthetic eukaryotes. It is also useful for studying the fundamental transcriptional network, as it possesses a small number of transcription factors, less than 100 for the 16.5 Mbp of the nuclear genome (Kuroiwa, 1998; Matsuzaki et al, 2004; Nozaki et al, 2007). With respect to TFIIB and related proteins, the C. merolae genome contains genes for TFIIB, BRF and pBrp, each as a single copy (see Results for details).

In this study, as a first step to understand the function of pBrp for nuclear transcription, we analysed its target gene in C. merolae by chromatin immunoprecipitation (ChIP) assays. Our data indicated that pBrp specifically occupies the rDNA promoter region in vivo, and in vivo and in vitro analyses showed that C. merolae pBrp is positively involved in the RNA polymerase I (Pol I)-dependent rRNA synthesis in the nucleolus. Moreover, we also show evidence that the pBrp function is also conserved for pBrp in A. thaliana. Thus, we propose here that pBrp is a GTF for Pol I in plant cells.

Results

PBRP gene in C. merolae and its expression

The C. merolae 100%-complete genome sequence (Matsuzaki et al, 2004; Nozaki et al, 2007) revealed 4775 open reading frames on 20 chromosomes and showed that CMI217C (gene number in http://merolae.biol.s.u-tokyo.ac.jp/), CML077C and CMA019C encode TFIIB-related proteins. To assess phylogenetic relationships among these genes, a maximum-likelihood tree was constructed with TFIIB and related proteins from yeast, human, green alga, higher plants and Archaea (Figure 1A). The resultant phylogenetic tree consists of four independent groups, each represented by eukaryotic TFIIB, BRF, pBrp and archaeal TFB, and showed that CMI217C, CML077C and CMA019C proteins were assigned into the pBrp, TFIIB and BRF groups, respectively. Thus, we designated here CMI217C, CML077C and CMA019C as CmpBrp, CmTFIIB and CmBRF, respectively. Comparison of the deduced amino-acid sequence of CmpBrp with those of higher plants' pBrps showed that CmpBrp has a higher molecular mass (981 aa, 104 kDa) and includes extensions on both amino- and carboxy-termini and several insertions without similarity (Supplementary Figure S1). However, the imperfect direct repeats exhibit a high degree of amino-acid sequence similarity to higher plants' pBrp proteins (Figure 1B and Supplementary Figure S1). The consensus TFIIB zinc-ribbon motif (Cys-X2-Cys/His-X15−17-Cys-X2-Cys) was observed in the higher plants' pBrp but not in CmpBrp, whereas a potential zinc-binding motif (Cys-X2-Cys-X19-Cys-X2-His) was found at the amino-terminal region of CmpBrp (Figure 1B and Supplementary Figure S1).

Figure 1.

Figure 1

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TFIIB-related protein family in C. merolae. (A) Evolutionary relationship of TFIIB-related proteins. Numbers at each node represent the percentage of trees supporting the specific branching pattern in the bootstrap analysis. Branch lengths are proportional to the number of amino-acid substitutions, indicated by the scale bar below the tree. Designations and GenBank accession numbers for sequences of the TFIIB-related proteins are shown in Supplementary data. MmTFB, PtTFB, HlTFB and NpTFB were used as the out-group to root the tree. (B) Schematic representation of secondary structure of CmpBrp, CmTFIIB and CmBRF. White, grey, black and hatched boxes indicate predicted putative zinc ribbon, B-finger, imperfect direct repeats and BRF domain, respectively.

To investigate CmpBrp expression, a rabbit polyclonal antibody against recombinant CmpBrp expressed in Escherichia coli was produced. The prepared antibody specifically recognizes endogenous CmpBrp protein (approximately 118 kDa, Supplementary Figure S2). We performed an immunoblot analysis using the CmpBrp antibody to probe C. merolae total protein extracted from cells exposed to several environmental stress conditions: high pH, high osmotic pressure, high temperature, high intensity of light, nitrogen starvation and carbon starvation. The results revealed that CmpBrp protein is constitutively expressed in the cell (data not shown).

Identification of CmpBrp target gene by ChIP

As pBrp is paralogous to TFIIB and BRF, we hypothesized that pBrp also functions as a GTF for nuclear RNA polymerase(s). To examine this possibility, we conducted ChIP analysis to identify the target gene in the nuclear genome, as a targeted gene disruption system for C. merolae has been not available to date. ChIP analysis was first carried out for proximal regions close to transcriptional start points (TSPs) of genes that are transcribed by the three classes of RNA polymerase; Pol I, Pol II and Pol III. Promoter regions of rDNA and 5S rDNA were representatively analysed as Pol I- and Pol III-dependent promoters, respectively. As for Pol II-dependent promoters, genes that show light-responsive expression patterns, that are light and dark responsive, that are constitutively expressed irrespective of the light conditions, or that are nitrogen-deprivation responsive were selected on the basis on microarray results (Kanesaki et al, our unpublished data; Figure 3A). Whereas TSPs of the Pol II-type genes could be predicted on the basis of the full-length EST analysis (Matsuzaki et al, 2004), those of the Pol I- and Pol III-dependent genes had not been experimentally identified. Therefore, these TSPs were first mapped at low resolution by northern blot analyses with several DNA probes (Figure 2A and D). The result indicated that the TSP of rDNA is located about 2000-bp upstream of the mature 5′-end (Figure 2B). Although this information was sufficient for ChIP analysis, the precise TSP of rDNA was further analysed by primer extension and S1 nuclease protection assays (Figure 2C) for in vitro biochemical analyses (Figures 4 and 5). It should be noted that the C. merolae nuclear genome encodes only three rRNA units at different chromosomal loci, named here as RRNa (CMQ305R), RRNb (CMQ401R) and RRNc (CMR208R) (Maruyama et al, 2004; Matsuzaki et al, 2004; Nozaki et al, 2007). Each of the three rDNA units in the coding and the upstream regions (up to ∼3.7-kb upstream from the putative 5′-end of 18S rRNA, http://merolae.biol.s.u-tokyo.ac.jp/) share an almost identical DNA sequence. Therefore, it is difficult to distinguish the major reverse transcription product and to determine which RRN gene(s) yielded the S1 nuclease-protected RNA. Providing that all rDNA genes are active, TSPs of rDNA genes were mapped to be 1949-bp upstream from the putative 5′-end of 18S rRNA for RRNa, 1949-bp for RRNb and 1946-bp for RRNc (Figure 2C and F). It is of note that the different TSP is due to some gaps between the mature 5′-end and TSP of the three rDNA genes. In any case, the TSP information is consistent with the data obtained by northern blot analysis (Figure 2B) and was sufficient for in vitro biochemical analyses shown in Figures 4 and 5. As for the 5S rRNA genes, there are three chromosomal loci; CML038R, CML060R and CMM232R. Nucleotide sequences around these three genes are almost identical, and the TSPs of 5S rRNA genes were mapped by northern blot analysis at almost identical positions in the mature 5′-end (Figure 2E). Thus, we did not analyse further to discriminate the three copies because the present information was sufficient for the ChIP analysis.

Figure 2.

Figure 2

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Determination of transcription start sites of C. merolae rDNA and 5S rDNA. (A) Positions of probes (a–f) used in northern blot analysis for rDNA. (B) Mapping of TSP of rDNA by northern blot analysis. C. merolae total RNA (6 μg) prepared from logarithmic-growth cells (approximately OD750=0.5) under continuous light conditions was subjected to northern blot analysis with relevant specific probes shown in panel (A). Transcript size is indicated at the left in kilonucleotides. Putative positions of prematured full-length rRNA (full length), prematured 18S rRNA (Pre-18S) and matured 18S rRNA (Matured 18S) are indicated at the right. Lower panel shows rRNA stained with methylene blue as a loading control. (C) 5′-end mapping of rDNA TSP by primer extension and S1 nuclease protection assays. Positions of primer (arrowhead) and DNA probe (line) used in primer extension analysis and S1 nuclease protection assay are indicated at the top. Asterisks indicate 32P labelled 5′-end of DNA. Primer extension analysis (left) or S1 nuclease protection assay (right) was carried out with the same RNA used in panel B (lane 1) or yeast RNA (lane 2). Arrowhead indicates the TSP of rDNA. (D) Positions of probes (a–e) used in northern blot analysis for 5S rDNA. (E) Mapping of TSP of 5S rDNA by northern blot analysis. Others are the same as in panel B. (F) Sequence of rDNA (RRNb) promoter region. Arrow denotes the TSP.

Cells were grown under continuous light conditions, and ChIP analysis with the CmpBrp antibody was performed as shown in Figure 3A. Intriguingly, the result indicated that CmpBrp occupies the promoter region of the rDNA (rDNA no. 1). The occupancy was not detected at the promoter regions of mRNA-encoding genes and 5S rDNA and was significantly reduced at approximately +2500 bp from the TSP of the rDNA (rDNA no. 2). These results implied that CmpBrp takes part in rRNA synthesis. To further understand this, we next examined the correlation between the occupancy of CmpBrp on the rDNA promoter region and the de novo 18S rRNA synthesis rate, which was determined by run-on transcription assay. By exposing to light after adaptation to darkness, the level of de novo 18S rRNA synthesis was apparently elevated approximately 2.0- to 2.3-fold that at L0 (just before the light irradiation). Upon re-exposure of the cells to darkness, the de novo 18S rRNA synthesis level dropped back to the level observed at L0. Under the same conditions as the run-on analysis, ChIP analysis was carried out and the result clearly indicated that levels of CmpBrp occupancies on the rDNA promoter region were well correlated with those of de novo synthesis of 18S rRNA (Figure 3B and D). The protein level of CmpBrp was constant irrespective of the light conditions (Figure 3C), suggesting that post-translational modification and/or other regulation of CmpBrp enhances its binding affinity to the rDNA promoter region.

Figure 3.

Figure 3

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CmpBrp specifically binds to rDNA promoter region in vivo. (A) Identification of CmpBrp target gene. ChIP analysis was performed with CmpBrp antibody (CmpBrp Ab.) or rabbit IgG purified from preimmune serum (Pre) using fixed logarithmic growth cells (OD750=0.5) under continuous light conditions. Occupancy of CmpBrp was measured at the indicated promoter regions. For rDNA, the ≈2500-bp downstream region from its TSP (rDNA no. 2) as well as its promoter region (rDNA no. 1) were analysed. Expression types of Pol II-type genes are shown at the bottom. L, light-responsive; L & D, light- & dark-responsive; Cont., constant; -N, N deprivation-responsive. Values are averages of at least three independent experiments and represent percent recovery relative to the total input DNA. Error bars indicate standard deviation. (B) Level of de novo 18S rRNA synthesis under the light/dark cycle. C. merolae cells were grown under white light until OD750=0.5 and incubated in complete darkness for 18 h. Lights were turned on for 6 h and then turned off again. Cells were sequentially harvested at the time (h) shown at the top, and the newly synthesized 18S rRNA was detected by run-on analysis. Signals obtained with rDNA (18S rRNA) or pDEST-HIS (negative control) as probe DNAs are shown in the middle. Signal intensities of de novo synthesized 18S rRNA from three independent experiments were quantified and values (L0 as 100%) are presented (means±s.d.) as relative levels at the bottom. (C) Protein level of CmpBrp in the light/dark cycle. Aliquots of total protein (15 μg) from the cell lysate were subjected to immunoblot analysis with CmpBrp antibody. Total protein (around 55 kDa) stained with Coomassie Brilliant Blue (CBB) is shown as loading control (lower panel). (D) CmpBrp association on the rDNA promoter region in the light/dark cycle. ChIP analysis was carried out in the same way as in panel (A) but with sample prepared similar to panel (B). Occupancy of CmpBrp at promoter regions of rDNA (rDNA no. 1) and CMK028C (K028) was analysed. (E, F) ChIP analysis with CmTFIIB (E) and CmBRF (F) antibodies. ChIP analyses were carried out with sample prepared at L2. Occupancies of relevant factors were analysed at the indicated promoter regions. Others are the same as in panel (A).

Next, we investigated a functional distinction among CmpBrp, CmTFIIB and CmBRF on the three types of RNA polymerase-dependent transcription. To achieve this, polyclonal antibodies against recombinant CmTFIIB and CmBRF were prepared (Supplementary Figure S2) and subjected to ChIP analysis with the sample prepared at L2. As in other eukaryotes, specific occupancies of CmTFIIB and CmBRF were detected at promoter regions of genes for mRNAs (CMJ289C and CMK028C) and 5S rRNA, respectively, whereas little occupancy was detected for the rDNA promoter region using both antibodies (Figure 3E and F). These results strongly suggested that CmpBrp is a specific GTF for rDNA transcription.

Cooperative binding of CmpBrp and CmTBP to the rDNA promoter region in vitro

To verify whether CmpBrp is actually able to bind directly to the rDNA promoter region, electrophoretic mobility shift assay (EMSA) analysis was performed. Recombinant CmpBrp and CmTBP proteins (Figure 4A) were subjected to EMSA, as one of the functional characteristics of TFIIB is the ability to bind TBP and form a stable ternary complex with promoters (Orphanides et al, 1996). Here, CmTFIIB was used as a control. Recombinant CmpBrp did not bind to the rDNA promoter region; however, further addition of CmTBP to the reaction mixture resulted in a stable ternary complex formation (Figure 4B, lane 3 versus 4). On the other hand, CmTFIIB did not form a complex with the rDNA promoter region even in the presence of CmTBP. To further examine the binding characteristic of CmpBrp, EMSA was also performed with the Ad2ML promoter, which is dependent on Pol II for transcription, and harbours canonical BRE and TATA-box consensus sequences. As shown in Figure 4C, a ternary complex was observed only with CmTFIIB but not with CmpBrp even in the presence of CmTBP (lane 4 versus 5), suggesting that the DNA recognition specificity of CmpBrp is different from that of CmTFIIB. The binding site of the CmpBrp–CmTBP complex in the rDNA promoter region was investigated by competition experiments with unlabelled probes at positions indicated in Figure 4D. Excess unlabelled cold probe harbouring the rDNA promoter region (−161 to +55, +1 as the TSP) and no. 7 probe (+2 to +28) markedly competed for protein binding (lanes 1 versus 2 and 9), whereas other probes did not. To identify the CmpBrp–CmTBP complex binding site in the no. 7 probe, we next performed EMSA competition analysis using scanning mutagenized no. 7 probes. The sequences of the probes are shown in Figure 4E. Our results clearly indicated that five of nine unlabelled probes, no. 7_Mt3 to no. 7_Mt7, all of which were mutated at position +8 to +22, lost competitive activity for ternary complex formation (Figure 4E). Consistent with this, DNase I footprinting analyses also revealed that the CmpBrp–CmTBP complex protected the rDNA promoter region at +6 to +22 of the sense strand and at +8 to +22 of the antisense strand (Figure 4F). To determine whether the identified site was critical for ternary complex formation, EMSA analysis was carried out using a DNA probe with mutagenized sequences at +8 to +22. As shown in Figure 4G, the stable ternary complex was not observed when the mutated probe was used (lane 2 versus 4). These results clearly indicated that CmpBrp and CmTBP cooperatively bind the rDNA promoter region in vitro, and the binding sequences corresponding to +8 to +22 are indispensable for the stable ternary complex.

Figure 4.

Figure 4

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Cooperative binding of CmpBrp and CmTBP to the rDNA promoter region in vitro and identification of its site. (A) Purified recombinant CmTBP, CmpBrp and CmTFIIB. Proteins (0.2 μg each) were resolved on a 12.5% SDS–PAGE gel and stained with CBB. Positions of molecular size markers are indicated in kDa at left. (B) EMSA with the rDNA promoter region. Fragment containing promoter region of rDNA (−161 to +55, +1 as TSP) was incubated with (+) or without (−) indicated recombinant proteins. Arrowhead indicates specific ternary complex. (C) EMSA with the Ad2ML promoter region. Fragment containing promoter region of Ad2ML (−41 to +15) was incubated the same as in panel (B). (D) Search for CmpBrp–CmTBP binding region by EMSA competition analysis. EMSA analysis was performed under the same conditions as in panel (B), lane 4, without (−) or with indicated unlabelled probes at positions as shown at the top. Cold probe denotes unlabelled promoter region of rDNA (−161 to +55). (E) Identification of CmpBrp–CmTBP binding site by EMSA competition analysis. EMSA analysis was performed in the same way as in panel (D) without (−) or with indicated unlabelled probes that were mutagenized at 3-bp resolution, except for no. 7 probe. Sequences of mutagenized probes are shown at the top (lower characters indicate mutated nucleotides). (F) DNase I footprint analysis of rDNA promoter with CmpBrp–CmTBP binding site. The sense (top) and the antisense strands (bottom) were labelled at either end and used without (−) or with (+) CmpBrp and CmTBP in footprint reactions. Arrows and black boxes indicate the TSPs and the regions protected from DNase I digestion, respectively. (G) EMSA with the mutated rDNA promoter region within the CmpBrp–CmTBP binding site. Wild-type (Wt) or the mutated probe within the CmpBrp–CmTBP binding site (+8 to +22) (Mt) of the rDNA promoter region was incubated without (−) or with (+) CmpBrp and CmTBP.

Involvement of Pol I and CmpBrp in the rDNA promoter transcription

It is widely believed that the rDNA is transcribed by Pol I in eukaryotes, and it is natural to assume that this is also the case in C. merolae. To confirm this, we performed ChIP analysis with an antibody against CmRPA190 (Supplementary Figure S2), the largest subunit of Pol I, and found that the rDNA promoter region was specifically co-immunoprecipitated with CmRPA190 (Figure 5A). This result clearly indicated the involvement of Pol I in rRNA synthesis in C. merolae. Next, we examined whether CmpBrp and CmRPA190 coexisted in C. merolae cells by immunoprecipitation analysis. Our results indicated that endogenous CmRPA190 co-immunoprecipitated with CmpBrp and vice versa (Figure 5B, lanes 3 and 5). No co-immunoprecipitation of CmTFIIB and CmBRF with CmpBrp or CmRPA190 was observed, indicating that CmpBrp is a component of the general Pol I transcription machinery.

Figure 5.

Figure 5

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CmpBrp positively contributes to Pol I-dependent rDNA transcription. (A) ChIP analysis with CmRPA190 antibody. Others are the same as shown in Figure 3E. (B) Co-immunoprecipitation of endogenous CmpBrp and CmRPA190. Immunoprecipitation was performed with antibodies against CmpBrp, CmRPA190 or relevant IgG (Pre), and co-immunoprecipitation was analysed by immunoblot analysis with each indicated antibody. As a control, 15% of cell lysate (input) was directly subjected to immunoblot analysis. (C) Specific in vitro transcription from the rDNA promoter. In vitro transcription was performed with C. merolae crude cell lysate and/or pUC119-RRN-Wt as a template (+, presence; −, absence). Schematic representation of pUC119-RRN-Wt is shown at the top. Arrow indicates the TSP of rDNA, whereas arrowhead indicates primer used for the primer extension analysis. Transcripts from rDNA promoter are marked at the right of the autoradiogram with an arrowhead. (D) Effect of antibody on in vitro transcription from the rDNA promoter. In vitro transcription analysis was performed under the same conditions as in panel C, lane 3, with (+) or without (−) indicated antibodies. (E) In vitro transcription analysis with the mutated rDNA promoter region within the CmpBrp–CmTBP binding site. In vitro transcription was performed with C. merolae crude cell lysate and pUC119-RRN-Wt (Wt) or pUC119-RRN-Mt (Mt). Hatched box indicates mutagenized region.

Involvement of CmpBrp in rDNA transcription was further analysed by an in vitro transcription system using crude cell lysate prepared from C. merolae cells. When the rDNA promoter region was used for the assay as template DNA, an apparent transcript from the TSP of rDNA was detected (Figure 5C, lane 3). No band was observed in the absence of template DNA (lane 1) or cell lysate (lane 2), nor were any bands detected using the parental plasmid without the rDNA sequence (data not shown). These data indicated that the detected transcript was accurately derived from the input rDNA promoter. The in vitro transcription was not affected by addition of α-amanitin (Supplementary Figure S3), which is an inhibitor for Pol II (highly sensitive) and Pol III (slightly sensitive), to the reaction mixture. In vitro transcription was unaffected even at a high concentration of α-amanitin (250 μg/ml), at which concentration de novo syntheses of mRNA by Pol II (CMS045C and CMK028C) and 5S rRNA by Pol III were selectively inhibited (Supplementary Figure S3). Thus, we concluded that the rDNA in vitro transcription was dependent on Pol I. To examine whether CmpBrp is required for the Pol I-dependent transcription, we observed the effect of adding the CmpBrp antibody to the reaction. The result showed that the in vitro transcription was severely inhibited by addition of the CmpBrp antibody (Figure 5D, lane 1 versus 3). A similar inhibition was also observed by addition of CmRPA190 antibody (lane 1 versus 5). However, the reaction was not inhibited by addition of IgG purified from relevant preimmune serum, anti-CmTFIIB antibody, or anti-CmBRF antibody. Moreover, the in vitro transcripts almost disappeared when the DNA template with a mutated CmpBrp–CmTBP binding site was used (Figure 5E, lane 1 versus 2). Thus, these in vitro transcription experiments demonstrated that CmpBrp is indispensable for effective Pol I-dependent rDNA transcription, and the binding site of the CmpBrp–CmTBP complex is defined as an essential rDNA core promoter element.

Intracellular localization of CmpBrp

In eukaryotic cells, rRNA is synthesized in a specialized structure within the nucleus called the nucleolus. Pol I and its transcription-related factors have been consistently observed in the nucleolus area, in which rRNA transcription is highly active (Grummt, 2003). Thus, we examined the intracellular localization of CmpBrp by indirect immunofluorescence microscopy analysis. A yellow–green fluorescence showing the CmpBrp signal was observed from a limited area in the nucleus (Figure 6A, top). On the other hand, signals of CmTFIIB and CmBRF were detected throughout most areas of the nucleus (Figure 6A, middle and bottom), as in the case of A. thaliana TFIIB2 (Koroleva et al, 2005). These results suggested that the majority of CmpBrp protein is located in the nucleolus. To test this hypothesis, we performed co-immunolabelling with CmpBrp and CmRPA190 antibodies. The results shown in Figure 6B clearly indicated that CmpBrp (red signal) co-localized with CmRPA190 (yellow–green). Under the conditions for detection of the Alexa Fluor 568-conjugated goat anti-rabbit IgG (for CmpBrp), chlorophyll fluorescence is detected as a red signal. Thus, there are two possible origins of the red signal: either from CmpBrp localization or from chlorophyll fluorescence. However, in panel A, the yellow–green signal showing CmpBrp was not observed in the chloroplast, indicating that the red signal detected in the chloroplast was derived from chlorophyll fluorescence. Therefore, these results suggested that CmpBrp localizes in the nucleolus and again indicate involvement in Pol I-dependent rDNA transcription.

Figure 6.

Figure 6

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Intracellular localization of CmpBrp. (A) Localization of CmpBrp, CmTFIIB and CmBRF. Fixed cells at L2 condition were first incubated with rabbit CmpBrp antibody (top), guinea pig CmTFIIB (middle) or CmBRF (bottom) antibodies, and the localizations were detected with Alexa Fluor 488-conjugated goat anti-rabbit IgG or anti-guinea pig IgG antibodies (yellow–green signal, immunostained). DAPI staining of cells (DAPI), merged image of immunostained and the nucleus DNA fluorescence (Merge), and differential interference contrast image (DIC) are shown. Positions of nucleus (n) and chloroplast (c) are indicated with arrowheads. Bar=2 μm. (B) Co-immunolabelling with CmpBrp and CmRPA190 antibodies. Fixed cells were first incubated with rabbit CmpBrp and guinea pig CmRPA190 antibodies simultaneously. Localization of CmpBrp was detected with Alexa Fluor 568-conjugated goat anti-rabbit IgG (red signal, CmpBrp) antibody, and CmRPA190 was detected with Alexa Fluor 488-conjugated goat anti-guinea pig IgG (yellow–green signal, CmRPA190) antibody. Merged images show immunostaining with CmpBrp and CmRPA190 antibodies (Merge). Asterisks indicate signal due to chlorophyll fluorescence. No. 1 and no. 2 denote independent cells. Others are the same as in panel (A).

Comparative studies of Arabidopsis pBrp

As mentioned above, in vivo and in vitro lines of evidence clearly indicated that pBrp is a GTF of Pol I for rDNA transcription in C. merolae. As pBrp is also found in higher plant lineages, we questioned whether this function is common among plant lineages. We produced a polyclonal antibody against AtpBrp, and performed ChIP analysis to examine whether AtpBrp occupies an rDNA promoter region in the nucleus. In this study, we used Arabidopsis T87 suspension-cultured cells, in which rRNA could be actively synthesized because of the rapid growth rate. Consistent with a previous observation (Lagrange et al, 2003), antibodies raised against recombinant AtpBrp specifically recognized 52 kDa proteins in Arabidopsis T87 cells (Supplementary Figure S2). As shown in Figure 7A, results of ChIP analysis using cells grown in continuous light conditions indicated that AtpBrp occupancy was observed on the promoter region of rDNA (Doelling and Pikaard, 1995), but not on Pol II-dependent (RBCS-1A, CAB2, GS1, L19 and L23A) and Pol III-dependent (tRNAGlu(CTG) and tRNALeu(AAG)) promoters (Hasegawa et al, 2003; Bertrand et al, 2005). In addition to ChIP analysis, EMSA analysis was performed to verify that AtpBrp can bind directly to the rDNA promoter region. Figure 7B shows SDS–PAGE analysis of recombinant proteins used for the EMSA analysis: AtpBrp, trigger factor (TF) and AtTBP2. TF is a protein tag for solubilization of the recombinant protein and was used as a control for TF-fused AtpBrp. Recombinant AtpBrp did not bind to the A. thaliana rDNA promoter region, whereas it formed a stable ternary complex in the presence of AtTBP2 (Figure 7C, lane 4 versus 6). On the other hand, the shift band was not observed with the TF tag even in the presence of AtTBP2 (lane 5). In addition to the strong specific shift band, a smeared signal was observed when AtpBrp was added to the reaction mixture (lanes 4 and 6). The signal seems to be derived from an unstable interaction between AtpBrp and the rDNA probe, as the smeared signal was not detected with TF alone (lane 3). These results indicated that recombinant AtpBrp and AtTBP2 cooperatively form a stable ternary complex with the rDNA promoter region in vitro. The binding site of the AtpBrp–AtTBP2 complex in the rDNA promoter region was investigated by competition experiments as in the case of CmpBrp (Figure 4D). The results indicated that probes nos. 2, 4, 5 and 7–9 strongly competed for the protein binding (Figure 7D). This indicated the presence of several binding sites for the AtpBrp–AtTBP2 complex on the rDNA promoter region. Together, these comparative in vivo and in vitro experiments strongly suggested that AtpBrp is also involved in Pol I-dependent rRNA synthesis in A. thaliana.

Figure 7.

Figure 7

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Comparative studies of Arabidopsis pBrp. (A) Occupancy of AtpBrp on regions of Pol I-, Pol II- and Pol III-type promoters in vivo. ChIP analysis was performed with AtpBrp antibody (AtpBrp Ab.) or guinea pig preimmune serum (Pre) using fixed logarithmic growth cells under continuous light conditions. Occupancy of AtpBrp was analysed at promoter regions of rDNA, RBCS-1A (RBCS), CAB2, GS1, 60S ribosomal protein L19 (L19) and L23A (L23), tDNAGln(CTG) (Gln) and tDNALeu(AAG) (Leu). Others are the same as in Figure 3A. (B) Purified recombinant AtpBrp, TF-tag and AtTBP2. Others are the same as in Figure 4A. (C) EMSA with the Arabidopsis rDNA promoter region. Fragment containing promoter region of the rDNA (−164 to +106, +1 as TSP) was incubated with (+) or without (−) indicated recombinant proteins. Arrowhead indicates specific ternary complex. (D) Competition EMSA. EMSA analysis was performed under the same conditions as shown in panel C, lane 6, without (−) or with indicated unlabelled probes (positions shown at the top). Cold probe denotes unlabelled promoter region of rDNA promoter region (−164 to +106).

Discussion

In this study, we have clarified the intrinsic function of the third type of TFIIB-related protein, pBrp, in plant lineages. CmpBrp was shown to specifically occupy the rDNA promoter region in vivo by ChIP analysis (Figure 3), and further in vivo and in vitro experiments led us to conclude that CmpBrp positively contributes to Pol I-dependent rRNA synthesis in the nucleolus (Figures 3, 4, 5 and 6). We also demonstrated that A. thaliana pBrp specifically associates with the A. thaliana rDNA promoter region in vivo and in vitro (Figure 7). A previous study reported that the transcript of the AtPBRP gene is abundant in root and flower buds, but not in leaves, stems and siliques (Lagrange et al, 2003). The organs in which there is active transcription coincide well with those that express AtTOR (Menand et al, 2002), the product of which has a critical role in rRNA synthesis in response to various environmental cues (Mayer and Grummt, 2006). These findings strongly support the view that pBrp is involved in rRNA synthesis in higher plants. Taking all these results into consideration, we concluded that pBrp is a GTF for Pol I in plant cells.

Pol I transcription machineries

The Pol I transcription machineries have been identified in yeast and mammals and have been characterized in detail by biochemical and genetic methods (Nomura, 2001; Grummt, 2003; Russell and Zomerdijk, 2006). These machineries are comprised of three main components for rDNA transcription: the Pol I enzyme and its associated factors, the TBP–TAFI complex that directly binds to the core promoter and the upstream regulatory element-binding complex. Comparison of these components between yeast and mammals revealed that only Pol I components are well conserved, although the TBP–TAFI complex is also essential for basal levels of rDNA transcription (Supplementary Tables S1 to S4) (Comai et al, 1992, 1994; Nomura, 2001; Friedrich et al, 2005). In plants, only genes for TBP and histones H3 and H4 were identified as fundamental proteins for rDNA transcription on the basis of the genome information of C. merolae and A. thaliana, whereas no other component for the upstream and core promoter-binding complex was identified (Supplementary Tables S1 and S2). Two Pol I-associated factors, RRN3 and PAF67, were found (Supplementary Table S3). However, 3 of 14 Pol I components of yeast were not found in C. merolae and A. thaliana (Supplementary Table S4). One of these three, RPA43, is essential for Saccharomyces cerevisiae viability and has an important function in the PIC assembly in yeasts and mammals (Peyroche et al, 2000; Miller et al, 2001). These findings strongly indicate the extensive divergence of Pol I transcription machineries among eukaryotes.

This study demonstrated that pBrp of plant cells requires TBP to form the stable ternary complex with the rDNA promoter region, in spite of the absence of the consensus TATA-box element (Figures 4B and 7C, and Supplementary Figure S4). It is of note that the TATA-box sequence at the TSP of A. thaliana rDNA (Supplementary Figure S4) has an important function in promoter strength and start-site selection, and serves a function other than TBP binding (Doelling and Pikaard, 1996). On the other hand, the SL1/TIF–IB/CF complex recognizes and binds to the core promoter element, in which the consensus TATA-box element is absent, through its TAFI proteins, and TBP is highly conserved in these complexes (Supplementary Table S1) (Comai et al, 1992, 1994; Nomura, 2001). Thus, it seems plausible that pBrp and TBP are components of a complex that binds to the core promoter, and the complex is a functional equivalent of the SL1/TIF–IB/CF complex in plant cells. This assumption is supported by the following results: (i) the in vitro Pol I-dependent rDNA transcription was severely inhibited by addition of the CmpBrp antibody (Figure 5D) and (ii) the binding sequence of the CmpBrp–CmTBP complex (+8 to +22) is indispensable for effective in vitro transcription (Figure 5E).

It was shown that TFIIB and BRF bind the core promoter elements, which are located at the proximal upstream sites of their target gene's TSP (Lagrange et al, 1998; Kassavetis and Geiduschek, 2006; Deng and Roberts, 2005, 2007) and have important functions in the transcription during the initiation processes (see Introduction). In A. thaliana, a previous study reported that the 5′-boundary of the rRNA gene promoter is located between −55 and −33 and the 3′-promoter boundary is at approximately +6 (Doelling and Pikaard, 1995). Consistently, several binding sites of the AtpBrp–AtTBP2 complex were identified in this study, and some of them were located within the core promoter region (no. 4 (−73 to −44) and no. 5 (−43 to −14) in Figure 7D). In contrast, in C. merolae, the binding site of CmpBrp–CmTBP was uniquely identified at sequences from +8 to +22 with respect to the TSP (Figure 4). The difference between C. merolae and A. thaliana could be attributed to the previously mentioned structural difference of their pBrp proteins, especially in the imperfect inverted repeats (Supplementary Figure S1), which are thought to be the DNA-binding domains as in TFIIB (see Introduction). However, the downstream regions of the TSPs appear to be important in both cases, because the downstream sequences of the TSPs showed high similarity to each other (Supplementary Figure S4), and it was observed that the AtpBrp–AtTBP2 complex also binds to the downstream region of the TSP (Figure 7D). This similarity was also observed between C. merolae and other higher plants, including Cucumis sativus, Pisum sativum, Raphanus sativus and Solanum lycopersicum (data not shown). The structural difference may also result in different binding specificity to the Ad2ML promoter between C. merolae and A. thaliana (Figures 4C and Lagrange et al, 2003). Further detailed study examining pBrp function will clarify the mechanism of Pol I-dependent rDNA transcription not only in C. merolae but also in higher plants.

pBrp evolved early in eukaryotic cell evolution and is conserved only in plant lineages

A major question yet to be addressed is why the Pol I-type TFIIB-related protein, pBrp, is found only in plants. It is generally accepted that eukaryotes and Archaea evolved from a common ancestor, which possessed a unique RNA polymerase similar to that of the Archaea (Kwapisz et al., 2008). The archaeal RNA polymerase is most similar to eukaryotic Pol II but requires the support of only two GTFs, TBP and TFB (archaeal homologue of TFIIB) to initiate basal transcription (Geiduschek and Ouhammouch, 2005). During subsequent evolution, eukaryotes have acquired three different types of RNA polymerase, Pol I–III, and the GTFs for each. Among them, TFIIB and its related protein, BRF, have evolved for Pol II- and Pol III-dependent transcription, respectively, whereas TBP has been used continuously for all RNA polymerases. Thus, it is natural to assume that Pol I-dependent transcription also required a TFIIB-related protein for transcription initiation, at least during the early phase of evolution. Consistently, the phylogenetic analysis shown in Figure 1A indicates that pBrp (Pol I-type), TFIIB (Pol II-type) and BRF (Pol III-type) have branched independently, and pBrp is not a descendant of TFIIB or BRF as expected from later horizontal transfer(s) from other eukaryotic lineages. Further genome information of divergent eukaryotic organisms could further clarify the evolutional story of pBrp.

Pol I transcription machineries are quite divergent among eukaryotes (see above) and tight species specificities of rRNA gene transcription have been reported (Doelling and Pikaard, 1996; Eberhard and Grummt, 1996). Thus, this high evolutionary speed could be a reason for the loss of Pol I-type pBrp in most eukaryotic lineages other than plants. Some requirements that are only required in plant cells, such as communication between the nucleus and plastid, might have been a restriction to prevent the subsequent loss of pBrp. Such a possibility could be discussed when the relevant function of plants' pBrp is elucidated in future studies.

Materials and methods

Strain and growth conditions

Cyanidioschyzon merolae 10D was grown at 42 °C under continuous white light (100 μE m−2 s−1) in liquid MA2 medium (Ohnuma et al, 2008) at pH 2.5 bubbling with air supplemented with 2% CO2. A suspension culture of the Arabidopsis (Arabidopsis thaliana (L.) Heynh., ecotype Columbia) cell line T87 established by Axelos et al (1992) was obtained from The RIKEN Bioresource Center (Tsukuba, Japan), and was maintained according to their instructions. Briefly, an aliquot (10 ml) of 1-week-old cells was subcultured into 75 ml fresh medium and grown under continuous white light (50 μE m−2 s−1) at 23 °C on a rotary shaker (100 r.p.m.). Samples for immunoblot and ChIP analyses were prepared from 3-day-old cell cultures.

Phylogenetic analysis

A maximum-likelihood tree based on 219 unambiguously aligned amino-acid positions of 19 TFIIB-related proteins was constructed as described previously (Osanai and Tanaka, 2007).

Preparation and purification of polyclonal antibodies

Preparation and purification of polyclonal antibodies were carried out as described previously (Imamura et al, 2003). See Supplementary data for details.

Immunoblot analysis

Immunoblot analysis was performed as described previously (Imamura et al, 2003) with slight modifications. See Supplementary data for details.

RNA preparation and analysis

Total RNA preparation and northern blot analysis were performed as described previously (Osanai et al, 2005). Primer extension analysis was carried out as described previously (Imamura et al, 2003). S1 nuclease protection assay was performed as described previously (Doelling and Pikaard, 1995) with modifications. See Supplementary data for details.

ChIP analysis

The detailed protocol for ChIP analysis is provided in Supplementary data.

Nuclear run-on transcription analysis

Isolation of nuclei and nuclear run-on reactions were carried out as described previously (Meininghaus et al, 2000), with slight modifications. See Supplementary data for details.

EMSA analysis

Reactions (20 μl) were carried out in 12 mM HEPES–KOH, 50 mM KCl, 3.5 mM MgCl2, 15% glycerol, 1 mM DTT, 0.2 μg poly(dG-dC)·poly(dG-dC) (Amersham Biosciences) as a non-specific competitor, 2.5 nM labelled probe and/or 100 nM of purified recombinant protein(s). Competition experiments were performed by adding oligonucleotides at 200-fold molar excess to the labelled probe. Detailed protocols for the preparation of recombinant proteins and DNA fragments used for EMSA analysis are provided in Supplementary data.

DNase I footprinting analysis

The detailed protocol for DNase I footprinting analysis is provided in Supplementary data.

In vitro transcription analysis

In vitro transcription analysis was performed as described previously (Schultz et al, 1991; Imamura et al., 2006). Detailed protocols for the preparation of template DNA and C. merolae crude cell lysate and in vitro reaction are provided in Supplementary data.

Immunoprecipitation analysis

The detailed protocol for immunoprecipitation analysis is provided in Supplementary data.

Indirect immunofluorescence microscopy analysis

Immunostaining with anti-CmpBrp, -CmTFIIB, -CmBRF and -CmRPA190 antibodies and DAPI staining were performed as described previously (Terashita et al, 2006) with some modifications (see Supplementary data).

Supplementary Material

Supplementary Figure S1

emboj2008151s1.tiff (837.3KB, tiff)

Supplementary Figure S2

emboj2008151s2.tiff (59.3KB, tiff)

Supplementary Figure S3

emboj2008151s3.tiff (35.1KB, tiff)

Supplementary Figure S4

emboj2008151s4.pdf (32.6KB, pdf)

Supplementary Data

emboj2008151s5.doc (190.5KB, doc)

Acknowledgments

We thank Dr Yu Kanesaki for communicating data before publication, Drs Tetsuro Kokubo and Hiromi Nishida for their technical advice and Ms Junko Nishida for her technical assistance. SI thanks Dr Ayaka Imamura for her encouraging support. This work was supported by a Grant-in-Aid for Creative Scientific Research (16GS0304 to KT) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. SI is supported by a research fellowship from the Japan Society for the Promotion of Science (JSPS) and a Grant-in-Aid for JSPS Fellows from the Ministry of Education, Science and Culture.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Figure S1

emboj2008151s1.tiff (837.3KB, tiff)

Supplementary Figure S2

emboj2008151s2.tiff (59.3KB, tiff)

Supplementary Figure S3

emboj2008151s3.tiff (35.1KB, tiff)

Supplementary Figure S4

emboj2008151s4.pdf (32.6KB, pdf)

Supplementary Data

emboj2008151s5.doc (190.5KB, doc)

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