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PLoS ONE. 2008; 3(12): e4072. Published online 2008 December 30. doi: 10.1371/journal.pone.0004072. | PMCID: PMC2605565 |
Copyright Reiner et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Function and Assembly of a Chromatin-Associated RNase P that Is Required for Efficient Transcription by RNA Polymerase I Robert Reiner, Natalie Krasnov-Yoeli, Yana Dehtiar, and Nayef Jarrous* Department of Molecular Biology, The Hebrew University-Hadassah Medical School, Jerusalem, Israel Laszlo Tora, Editor Institute of Genetics and Molecular and Cellular Biology, France Received September 11, 2008; Accepted December 1, 2008. Background Human RNase P has been initially described as a tRNA processing enzyme, consisting of H1 RNA and at least ten distinct protein subunits. Recent findings, however, indicate that this catalytic ribonucleoprotein is also required for transcription of small noncoding RNA genes by RNA polymerase III (Pol III). Notably, subunits of human RNase P are localized in the nucleolus, thus raising the possibility that this ribonucleoprotein complex is implicated in transcription of rRNA genes by Pol I. Methodology/Principal Findings By using biochemical and reverse genetic means we show here that human RNase P is required for efficient transcription of rDNA by Pol I. Thus, inactivation of RNase P by targeting its protein subunits for destruction by RNA interference or its H1 RNA moiety for specific cleavage causes marked reduction in transcription of rDNA by Pol I. However, RNase P restores Pol I transcription in a defined reconstitution system. Nuclear run on assays reveal that inactivation of RNase P reduces the level of nascent transcription by Pol I, and more considerably that of Pol III. Moreover, RNase P copurifies and associates with components of Pol I and its transcription factors and binds to chromatin of the promoter and coding region of rDNA. Strikingly, RNase P detaches from transcriptionally inactive rDNA in mitosis and reassociates with it at G1 phase through a dynamic and stepwise assembly process that is correlated with renewal of transcription. Conclusions/Significance Our findings reveal that RNase P activates transcription of rDNA by Pol I through a novel assembly process and that this catalytic ribonucleoprotein determines the transcription output of Pol I and Pol III, two functionally coordinated transcription machineries. Transcription is carried out by functionally distinct nuclear RNA polymerases (pols) associated with general transcription factors, as well as specificity and coregulatory factors that assist in formation and function of preinitiation complexes. Pol I transcribes rRNA genes, Pol II mainly synthesizes protein-coding genes, while Pol III transcribes a large set of small noncoding RNA genes. Recent findings reveal that noncoding RNAs associate with and regulate pols I, II and III [1]– [6]. Thus, U1 snRNA and 7SK RNA regulate initiation and elongation of transcription by Pol II [7], [8], Alu RNA represses transcription by binding to Pol II in response to heat shock [9], IGS RNA facilitates silencing of Pol I transcription of rRNA genes through interaction with the chromatin remodeling complex NoRC [3], while the H1 RNA subunit of human nuclear RNase P is required for Pol III transcription of small noncoding RNA genes [10], [11]. These noncoding RNAs act in the context of ribonucleoprotein complexes [3], [10], [12]. Human nuclear RNase P has been initially characterized as a tRNA processing ribonucleoprotein, consisting of H1 RNA and at least ten distinct protein subunits, termed Rpp14, Rpp20, Rpp21, Rpp25, Rpp29, Rpp30, Rpp38, Rpp40, hPop1 and hPop5 [13]. The endonucleolytic activity of human RNase P in tRNA processing requires its H1 RNA entity, which recognizes precursor tRNA as substrate [14]. A recent work reports that H1 RNA mediates cleavage of precursor tRNA in the absence of protein [15]. Accordingly, the main input of the numerous protein subunits of human RNase P should be in other complex and versatile tasks of this ribonucleoprotein complex, e. g. transcription [11], [16], as will be further corroborated in this study. We have previously demonstrated that human nuclear RNase P is required for transcription of small noncoding RNA genes transcribed by Pol III [10], [11]. RNase P exerts its role in transcription through association with Pol III and with chromatin of Pol III genes, including the 5S rRNA genes whose transcripts are not known to be processed by RNase P [10]. RNase P acts as an auxiliary factor for Pol III, as is the case with the transcription factors TFIIIA, TFIIIB and TFIIIC. This latter concept is based on the fact that Pol III can catalyze transcription reactions in a simplified in vitro transcription system in the absence of TFIIIB and TFIIIC that facilitate reinitation of transcription [17]. Moreover, Pol III requires only TFIIIB for transcription of tRNA and 5S rRNA genes in vitro [18], [19] and a highly purified human Pol III combined with recombinant SNAPc and TFIIIB subunits can direct multiple cycles of in vitro transcription initiation and termination from a U6 snRNA gene promoter [20]. H1 RNA is an abundant molecule in the cell. This transcript was detected in the cytoplasm, nucleoplasm and nucleoli. Protein subunits of human RNase P have also been differentially detected in specialized intranuclear compartments associated with active gene transcription, including nucleoli [16]. Mass spectrometry analysis of highly purified nucleoli of human cells confirms the existence of many protein subunits of RNase P, including Rpp14, Rpp20, Rpp25, Rpp29, Rpp30, Rpp38, Rpp40 and hPop1, in these bodies [21]. Indirect immunofluorescent analyses demarcate some of these protein subunits in confined sub-nucleolar sites, such as Rpp29 that resides in the dense fibrillar component, in which transcription and processing of rRNA take place [22], [23]. RNase P shares its protein subunits with the nucleolar ribonucleoprotein RNase MRP, except for the subunits Rpp21 and H1 RNA, which could be used to discriminate between the two ribonucleoproteins [24]. The exact role of human RNase MRP in the nucleolus remains unknown while its yeast counterpart is required for processing of 5.8S rRNA [25]. In this study, we show by biochemical and reverse genetic means that H1 RNA and its protein subunits, as part of an RNase P ribonucleoprotein, are required for efficient transcription of rDNA by Pol I. RNase P copurifies and associates with components of Pol I and its transcription initiation factors and exerts its role in transcription through association with the promoter and coding region of rDNA. Furthermore, we demonstrate that RNase P disengages from rDNA in mitosis and reassociates with it at G1 phase through a dynamic and stepwise assembly process. Our data implicate a catalytic ribonucleoprotein in transcription by Pol I and Pol III, whose coordinated function is critical for protein synthesis and cell growth. Knockdown of protein subunits of human RNase P inhibits Pol I function Rpp29 was targeted for destruction in HeLa cells by the use of RNA interference. Western blot analysis revealed efficient knockdown of Rpp29 in cells transfected with siRNA29 [26] but not with luciferase siRNA (). Transcription of a human mini-rDNA gene, which is composed of rDNA promoter and adjacent 5′ETS segment fused to a terminator region (), took place in the control whole cell extracts () but not in those prepared from cells treated with siRNA29 (; ). Similarly, transcription of a human 5S rRNA gene was deficient in the extracts derived from cells with knockdown of Rpp29 (), as previously shown [10]. Moreover, knockdown of Rpp29 by siRNA29 () caused a marked decrease in 45S pre-rRNA synthesis in cells, as determined by RT-PCR (). The primers used for detection of the pre-rRNA recognize the 5′ETS, which is processed quickly from the primary transcript during transcription and thus reflects transcription initiation by Pol I. By contrast, expression of the U1 snRNA, which is transcribed by Pol II, remained unchanged (). Inhibition of Pol I and Pol III transcription is not restricted to knockdown of Rpp29. Thus, knockdown of the subunit Rpp21, which is not shared with RNase MRP, by either siRNA or external guide sequence () [26] resulted in ~90% reduction in transcription of the mini-rRNA and 5S rRNA genes (). More importantly, nuclear run-on transcription assays confirmed that the level of nascent transcription of endogenous rRNA genes decreased by 5- and 10-fold in cells treated with siRNA21 and EGS Rpp21, when compared with control cells (). A more prominent reduction (~34-fold) in the level of nascent transcription of 5S rRNA gene by Pol III was measured (). Since this nascent transcription reflects the amount of active polymerases on the tested template, the results indicate that RNase P determines the transcription output by Pol I and Pol III. Reconstitution assays of Pol I transcription Knockdown of Rpp21, Rpp29 or Rpp38 by RNA interference has been shown to be accompanied by coordinate inhibition of expression of other protein subunits of human RNase P [26], [27; also data not shown]. This phenomenon made the development of an in vitro reconstitution system for Pol I and Pol III transcription using defined protein components of RNase P unfeasible. However, knockdown of the subunit Rpp25 by siRNA [26] led to moderate inhibition of expression of only two subunits, Rpp20 and Rpp21 (; and data not shown), while it resulted in severe inhibition of transcription of the mini-rRNA gene () and 5S rRNA gene (). Therefore, we checked if transcription in these extracts can be rescued by a histidine-tagged, recombinant Rpp25 protein that was highly purified through a chelating chromatography column (). Remarkably, the addition of recombinant Rpp25 (see Materials and Methods) to whole extracts obtained from cells with knockdown of Rpp25 resulted in reconstitution of transcription of the mini-rRNA and 5S rRNA genes, when compared with transcription in untreated extracts (). The gain-of-function of Pol I transcription was partial () while that of Pol III transcription was more prominent (; see below). The addition of recombinant Rpp25 to extracts of mock-transfected cells had no comparable consequences on transcription (). The addition of recombinant Rpp20 protein () had no effect on transcription (), while inclusion of recombinant Rpp20 and Rpp25 or recombinant Rpp20, Rpp21 and Rpp25 proteins rather reduced transcription (). Rpp20 and Rpp25 belong to the Alba-like superfamily of chromatin proteins [28] and it seems that the former protein neutralizes the effect of Rpp25 on transcription. Flow cytometry analysis revealed a decrease in the proliferation of the cells described above after 2 and 3 days in culture, in which cells reached high confluence (data not shown), and thus exhibited reduced activity of Pol I (). This reduction in Pol I activity may have counteracted the effect of recombinant Rpp25 in reconstitution assays. Therefore, we repeated the reconstitution experiment as described in but this time we included a 24 h time point, in which knockdown of Rpp25 was evident (). The results show that the addition of recombinant Rpp25 to the extract obtained at 24 h time point brought to almost full recovery of transcription of the mini-rRNA gene, when compared to transcription in extracts obtained at later time points (). The findings described above provide evidence that a protein subunit of human RNase P can substitute for its endogenous counterpart by reconstituting transcription of Pol I and Pol III. Protein subunits and H1 RNA are required for efficient Pol I transcription Immunodepletion of active RNase P from whole HeLa extracts by the use of polyclonal antibodies directed against Rpp20 and Rpp25 (see Materials and Methods) [10] resulted in reductions of ~40% and 80% in transcription of the mini-rRNA gene, which could be restored by adding back their corresponding immunoprecipitates (). Moreover, targeted cleavage of the H1 RNA moiety in whole HeLa extracts by RNase H and an antisense deoxyoligonucleotide H1-1, which is directed against the specificity domain of H1 RNA (), caused incorrect processing of the 5′ leader sequence of precursor tRNA Tyr by RNase P (; arrow head) and abolished transcription of the mini-rDNA gene (). By contrast, the use of a scrambled H1-1 oligonucleotide, H1-1sc, did not alter enzyme specificity () or transcription (). Similar results were obtained for transcription of the 5S rRNA gene (). Neither RNase H nor oligonucleotides alone had an effect on RNase P or transcription (data not shown; ref. 10). Since H1 RNA discriminates RNase P from RNase MRP, the results establish that the former ribonucleoprotein, containing an intact H1 RNA, is implicated in transcription by Pol I. RNase P copurifies and associates with components of Pol I and its transcription factors To check if RNase P is associated with Pol I, S100 extracts of HeLa cells were fractionated in a DEAE-Sepharose anion exchange chromatography column [13; see Materials and Methods] and the eluted fractions with the peak of RNase P activity were then purified in a hydrophilic Sephacryl S-100 gel filtration column [14]. The excluded fractions were assayed for RNase P activity in tRNA processing () and tested for the presence of Rpp29 and Rpp25 (; lower panels). Components of Pol I, such as RPA194 and RPA43, and its associated transcription factors, including the upstream transcription factor UBF and the TBP-associated factor TAF I110, were all detected in fractions F26-F30 enriched with RNase P (). Moreover, active RNase P is physically associated with RPA43, RPB8, UBF and TAF I110 in these fractions as revealed by coimmunoprecipitation experiments (). The above findings demonstrate that an active RNase P associates with components of Pol I, which seems to exist in a large complex with its transcription initiation factors. The purification of a large (M r>2000 kDa) murine Pol I holoenzyme with its transcription factors has been reported [29]. RNase P and Pol I are located at the promoter and coding region of rDNA To have a mechanistic explanation of how human RNase P acts with Pol I in rRNA gene transcription, we tested if the former multi-protein complex associates with rDNA in rapidly dividing HeLa cells by chromatin immunoprecipitation (ChIP) analysis (see Materials and Methods) [10]. Genes for rRNA are organized in tandem transcription units separated by intergenic spacers [30], [31] and each unit has 18S, 5.8S and 28S rRNA coding regions that are interspersed by internal and external transcribed spacers (). ChIP analysis revealed 5.8S rDNA in chromatin samples brought down by antibodies directed against Rpp20, Rpp21 (not shared with RNase MRP), Rpp29 and Rpp38 (, respectively), as well as RPB8 (), a core protein component of Pol I (also of Pol II and III). No PCR product was seen when preimmune rabbit serum or antibody directed against the tumor suppressor p53 were tested (). A human tRNA Tyr gene was detected in chromatin samples brought down by antibodies against Rpp20, Rpp21 and Rpp29 () but not Rpp38 (), consistent with previous findings [10]. The U1 snRNA gene, which is transcribed by Pol II, was enriched in chromatin precipitated by the antibody against RPB8 (), while the transcriptionally inactive ARPP P0 gene was not detected in any chromatin sample (). Chromatin occupancy of 5.8S rDNA by Rpp25 was almost eliminated in cycling HeLa cells transfected with siRNA25 for 48 h, when compared with cells treated with luciferase siRNA (), while that of Rpp20, Rpp29 and RPB8 showed no comparable decline (). Hence, binding of Rpp25 to rDNA loci is independent of that of other protein subunits of RNase P and Pol I. We next scanned the human rDNA transcription units (each is ~14 Kbp) for binding of protein subunits of RNase P by ChIP analysis using specific primers that correspond to distant regions within these units (see ). In these experiments, the genomic DNA was sheared to small pieces of ~500 bp to allow high resolution of chromatin binding regions (data not shown). Surprisingly, we found that Rpp20, Rpp21, Rpp29 and Rpp30 associated with the promoter region, in addition to the coding region spanning 18S rDNA and 28S rDNA (). Similarly, RPA194, a protein subunit of Pol I, bound to the promoter and coding region of rDNA (), consistent with previous findings reported by others [32]. RPA194 did not bind to the intergenic sequence located ~5 Kbp downstream of the terminator region of rDNA, which was also the case with the protein subunit of RNase P, except for Rpp29 that showed weak signal of binding (). This latter finding rules out the possibility that RNase P is distributed uniformly on rDNA repeats. Dissociation and reassociation of RNase P with chromatin of rDNA during the cell cycle Transcription of rRNA genes is regulated during the cell cycle, whereby it gradually increases from G1 to S phase, peaks at G2 and ceases at mitosis [33], [34]. To show that RNase P associates with transcriptionally active rRNA genes, HeLa cells were synchronized to G2/M by treatment with nocodazole, a microtubules depolymerization inhibitor, and then the separated G2 and M cell populations [35; see Materials and Methods] were subjected to flow cytometry () and ChIP analyses. Rpp21 and Rpp29 were associated with 5.8S rDNA and 18S rDNA in G2 cells but fully or partially disengaged from these loci in mitotic cells (, respectively). Moreover, chromatin occupancy of these pre-rRNA coding regions by RPB8, which represents Pol I, was evident in cells at G2 but not at M phase (), an observation that is consistent with cessation of transcription [34], [36]. In fact, all protein subunits of RNase P, including Rpp30, Rpp38 and Rpp40, were bound to rDNA in G2 cells but not in mitotic cells ( and data not shown). Western blot analysis showed that the expression of some protein subunits, such as Rpp29 and Rpp38, decreased in mitotic cells, when compared with G2 cells, while that of Rpp20 and Rpp40 remained largely unchanged (; see below). This implies that the detachment of these protein subunits from rDNA in mitosis is a shared property but their fate is not interrelated (see below). We next investigated the recruitment of protein subunits of RNase P after mitosis in synchronized HeLa cells. Strikingly, we found that Rpp20 and Rpp29 reassociated with 5.8S rDNA after 2 h of exit of synchronized HeLa cells from mitosis () but Rpp25 did not (). Similar pattern of chromatin occupancy by these three subunits was observed when the 5S rRNA gene was analyzed (; middle panel), while both Rpp29 and Rpp25 were not detected at the tRNA Leu gene until mid G1 (). Therefore, recruitment of Rpp20, Rpp25 and Rpp29 to chromatin is not interdependent. Of note, RPB6 promptly bound to 5.8S rDNA, 5S rRNA and tRNA Leu genes at early G1 () even though this binding does not reflect vigorous Pol I transcription, since it has been shown that UBF is inactivated at early G1 [33], [34]. Indeed, transcription of human tRNA Tyr and 5S rRNA genes was also impaired at early G1 () but gradually increased as the synchronized cells () progressed to S phase (). By contrast, binding of Rpp20 to chromatin of 5S rRNA and tRNA Leu genes somewhat decreased at S phase () The aforementioned observations raised the question if the biosynthesis of the protein subunits of human RNase P during the cell cycle correlates with chromatin binding by RNase P and with the transcriptional activity of Pol I and Pol III. Thus, the steady state levels of several protein subunits of RNase P were determined in synchronized HeLa cells that were released from hydroxyurea, which arrests cells at G1/S phase (). Expression of Rpp25 and Rpp29 increased during the S phase, peaked at G2, declined at mitosis and recovered at G1 (). This pattern agrees with the reported activity of Pol I and Pol III during the cell cycle [34], [35]. However, we found that the expression of Rpp20 and Rpp40 rather remained largely unchanged throughout the phases of the cell cycle, including mitosis (), in which these subunits also detached from chromatin (data not shown). Hence, the biosynthesis of these latter protein subunits does not reflect their dynamic binding to chromatin or transcription during the cell cycle. Taken together, the results described above indicate that restoration of Pol I and Pol III transcription after mitosis is not correlated with the recruitment of a fully preassembled RNase P on rDNA and small noncoding RNA genes. To the contrary, its assembly involves a dynamic and stepwise association process, which includes two Alba-like chromatin proteins, Rpp20 and Rpp25. We have shown that human RNase P is required for efficient transcription of rDNA by Pol I. Thus, inactivation of RNase P by targeting its protein and RNA subunits for destruction inhibits rRNA gene transcription by Pol I, an indication that this complex acts in the form of ribonucleoprotein. RNase P associates with components of Pol I and its transcription factors and affects nascent transcription by Pol I, as well as Pol III. RNase P binds to chromatin of the promoter and coding region of rRNA genes. Chromatin occupancy by RNase P is dependent on active transcription of rRNA genes and is linked to the cell cycle. Combined with the role of RNase P in Pol III transcription and binding to chromatin of 5S rRNA and tRNA genes [10], [11], the data presented in this study suggest that this chromatin-associated complex is critical for the coordinate regulation of ribosome biogenesis and protein translation. A novel role for RNase P in the nucleolus In S. cerevisiae, the majority of the RNase P RNA exists in the nucleolus, in which clustering of tRNA genes and processing of precursor tRNAs take place [37]. In situ RNA hybridization analysis also visualized H1 RNA in the nucleolus, thus raising the possibility that RNase P may be functional in ribosome biogenesis. Our study establishes that a functional human RNase P exists in the nucleolus and that it is implicated in transcription of rRNA genes. ChIP analysis demonstrates that many protein subunits of RNase P, including its subunit Rpp21 that is not shared with RNase MRP, bind to chromatin of transcriptionally active rDNA loci. This nucleolar form of RNase P is comparable, in terms of its protein composition, to that initially characterized as a tRNA processing holoenzyme [13]. In yeast, processing of rRNA is coordinated in a transcription-dependent manner [38]– [40]. Yeast Pol I associates with a nucleolar substructure that is active in the synthesis and processing of rRNA [41] and it has been demonstrated that transcription and processing of rRNA are coordinated through specific components of the small ribosomal subunit processome [38], [40], [42]. However, human RNase P does not act on transcription of rDNA as a processing factor. First, the mini-rRNA gene construct was designed to have a rDNA promoter fused to adjacent 5′ETS segment, which has no known cleavage sites that could recruit processing factors at the transcript level. Nonetheless, transcription driven by this promoter is inhibited in extracts lacking functional RNase P, as a result of depletion of its subunits, including H1 RNA and Rpp21 that are not shared with RNase MRP. In fact, it has been shown that transcription and processing factors are recruited separately in mammalian cells [39], [43]. Second, RNase P binds to the promoter region of rDNA and associates with components of the transcription initiation machinery, e. g. Pol I, UBF and SL1 complex, suggesting that it is involved in transcription initiation. Third, RT-PCR analysis of 5′ETS unveils a substantial reduction in pre-rRNA synthesis in cells having inactive RNase P. 5′ETS is known to be processed rapidly and cotranscriptionally from the primary transcript of rRNA and thus reflects transcription initiation by Pol I. Assembly of a chromatin-associated RNase P and its link to the cell cycle Binding of RNase P to chromatin of rDNA is dynamic in the sense that it is linked to transcription and the cell cycle. In mitosis, RNase P detaches from chromatin of rDNA, which concurs with cessation of transcription, while most of the protein subunits of RNase P were found to be associated with chromatin of rDNA at G2 phase, in which transcription by Pol I is elevated. At early G1, however, not all the protein subunits of RNase P are equally reassociated with chromatin of rDNA. For example, Rpp20 and Rpp29 but not Rpp25 are promptly recruited to rDNA after exit from mitosis, while Rpp25 binds to rDNA loci at the S phase (or late G1), which coincides with increased transcription by Pol I. Accordingly, Rpp25 seems to contribute to the elevated activity of Pol I at S phase (or late G1), a conclusion that is supported by the finding that recombinant Rpp25 stimulates transcription by Pol I in whole cell extracts lacking endogenous Rpp25. Similarly, recruitment of RNase P to 5S rRNA and tRNA genes after mitosis proceeds via a dynamic and stepwise association process of its subunits. A line of evidence supports the prospect that protein subunits of human RNase P could be recruited as independent entities to chromatin of target genes. Thus, RNA interference shows that knockdown of one protein subunit does not necessarily obstruct the recruitment of other subunits on target genes in cycling cells, even though transcription is inhibited. For example, knockdown of Rpp25 did not lead to disengagement of Rpp20 and Rpp29 from rDNA () and knockdown of Rpp29 did not affect chromatin occupancy of tRNA and 5S rRNA genes by Rpp21 [10]. Moreover, Rpp21 and Rpp29 differentially disengage from chromatin of tRNA and 5S rRNA genes in response to cessation of proliferation of cells [10]. Another research group showed that Rpp29 rapidly shuttles between the nucleoplasm and nucleolus, suggesting that this core component of RNase P is highly dynamic and not permanently bound to a fixed RNase P [44]. That RNase P is recruited as individual subunits (or partial sub-complexes) is consistent with the finding that subunits of Pol I rapidly and massively enter the nucleolus as distinct subunits rather than as part of a preassembled holoenzyme [45] and that the assembly of Pol I proceeds via a sequential manner in each transcription cycle [39], [45]. More importantly, it has been shown that the recruitment efficiency and retention of Pol I components at the promoter region of rDNA is used to control the transcriptional output of rRNA genes [46]. Apparently, these latter findings contrast the existence of assembled holoenzymes of Pol I [29] and RNase P [13]. Nonetheless, the purified Pol I-RNase P complex described in this study may constitute the fraction of these holoenzymes that have already been assembled and/or engaged in transcription. Nuclear run-on transcription assays indicate that the rate of nascent transcription of rDNA and 5S rRNA genes, which reflects the number of active Pol I and Pol III on these genes, is reduced in cells with inactivated RNase P. Thus, RNase P determines the output of transcription by active polymerases. Remarkably, ChIP analysis showed that chromatin occupancy by Pol I and Pol III (as represented by the core components RPB6 and RPB8) did not change perceptibly in cells with inactivated RNase P (; also refs. 10 and 11), an indication that these two polymerases can be recruited to their corresponding templates, even though there is no active transcription. Accordingly, RNase P defines those polymerases that are engaged in active transcription. However, the exact role of RNase P that is engaged in transcription remains unknown. This complex can act as auxiliary transcription factor, chromatin modifier or as new type of chromatin remodeling complex. In the latter case, it has been shown that RNase P has an ATPase activity embedded within its Rpp20 subunit [47] but this chromatin-associated subunit is not related to the SNF2-like ATPases. Nonetheless, binding of RNase P to the entire rDNA transcription unit () could also suggest that this complex has multiple roles in the various steps of transcription by Pol I. Finally, binding of a large ribonucleoprotein complex, such as human RNase P with its two Alba-like proteins, Rpp20 and Rpp25, to chromatin of hundreds of genes transcribed by Pol I and Pol III should have global effects on the structure, spatial organization and function of the genome [48]. As RNA can act as a scaffold, recruiting factor or sequence-specificity factor in the modification and function of chromatin [49], elucidation of any potential role of H1 RNA of RNase P in this regard is of particular interest. Cell transfection, synchronization and flow cytometry analysis Adherent HeLa cells were grown in high glucose DMEM (Invitrogen) supplemented with 5% fetal bovine serum, streptomycin (100 µg/ml), penicillin (100 U/ml) and Nystatin (12.5 U/ml). Cells were incubated in 5% CO 2 at 37°C. For transfection, 1–5×10 5 cells grown in 92×17 mm style petri dishes were transfected with the desired siRNA (15–30 µg/dish) or plasmid DNA (15 µg) in 10 ml medium using the calcium phosphate method. For knockdown of Rpp21 and Rpp25, plasmids (10–15 µg) carrying siRNA-coding genes (kind gifts of Prof. Sidney Altman) [26] were introduced into cells. For synchronization, HeLa cells were grown in 92×17 mm style petri dishes for 50% confluence and then treated with nocodazole (40 ng/ml) for 16 h. The dishes were shaken to detach mitotic cells from G2-enriched, adhered cells as described by White et al. 1995 [ref. 35]. Synchronization of cells by hydroxyurea (2.5 mM) was for 16 h. Cells were released from the inhibitor by replacing the medium with warm, fresh one. After time points indicated in each experiment the cells were then stained with propidium iodide for DNA content analysis by fluorescence-activated cell sorter. Whole cell extracts were prepared as described [10]. Protein concentrations in extracts were 10–15 mg/ml. RT-PCR analysis Reverse transcription was performed using M-MLV reverse transcriptase (Promega) with equal amounts of total RNA (1–2 µg) extracted from treated HeLa cells and 5–10 pmol of gene-specific reverse primers (see below). Amplification of the 5′ETS sequence of the reverse-transcribed pre-rRNA by PCR was performed as described by others [50] except that the reaction contained 5% Dimethyl sulfoxide (DMSO). Reverse transcription and PCR amplification of U1 snRNA was done using primers described previously [10]. Purification and analysis of human RNase P Rapidly dividing HeLa cells (at ~50% confluence) in sixty 175-cm square flasks were pelleted, disrupted, and the cell homogenate was centrifuged at 7,000 rpm followed by another centrifugation at 31,000 rpm in a Beckman Ti60 rotor to obtain S100 crude extract [13]. This S100 extract was loaded on a DEAE-Sepharose anion exchange chromatography column equilibrated with buffer A that contains 10 mM Tris-HCl, pH 8.0, 100 mM KCl, 2.5 mM MgCl 2 and 1 mM DTT. RNase P was then eluted from the column using a 100–500 mM KCl gradient [13]. RNase P activity in the eluted fractions was examined by processing of the 5′ leader of a 32P-labeled E. coli precursor tRNA Tyr. RNase P activity is eluted at 250–300 mM KCl [13], and therefore it coelutes with Pol I [51]. However, these fractions enriched with RNase P and Pol I are inactive in transcription of a mini-rRNA gene (data not shown), most likely because TIF-IA dissociates from Pol I in the presence of MgCl 2 [51], which was included in buffer A. Fractions enriched with active RNase P were then pooled, concentrated and fractionated in Sephacryl S-100 HR chromatography column with protein size markers [14]. Volumetric flow rate of the gel filtration column was set at ~0.8 ml/min. The eluted fractions were assayed for the presence of active RNase P as described above. RNase P eluted as a large particle from the column (with fractionation range of M r = 1×10 5) in the void volume. In vitro transcription of Pol I and Pol III and reconstitution assays In vitro transcription reactions of the mini-rRNA gene in newly prepared whole HeLa extracts were performed in a final volume of 25 µl that contained 15 µl of extract, 1× transcription buffer (12 mM Tris-HCl at pH 7.9, 5 mM MgCl 2, 80 mM KCl, 0.5 mM DTT, 10 mM creatine phosphate), NTPs (0.66 mM ATP, 0.66 mM CTP, 0.66 mM GTP, 0.01 mM UTP), 5 µCi of [α- 32P]-UTP (3000 Ci/mmol; Amersham) and 0.25 µg of plasmid carrying the mini-rDNA gene. After 1 h of incubation at 30°C, reaction mixtures were diluted 1 ![[ratio]](/corehtml/pmc/pmcents/x2236.gif) 1 with H 2O, passed through a G-50 column, diluted to 250 µl with 1× digestion buffer (20 mM Tris-HCl, pH 7.9, 250 mM sodium acetate, 1 mM EDTA, 0.25% SDS), and digested with 120 µg/ml Proteinase K for 30 min at 37°C. RNA was recovered following phenol chloroform isoamylalcohol extraction by ethanol precipitation and labeled RNAs were analyzed in 8% polyacrylamide gels. Bands were visualized by autoradiography and quantitated by Scion Image software. Transcription reactions with human 5S rRNA and tRNA Tyr genes were performed as previously described [10]. For reconstitution assays, recombinant proteins purified through His-bind affinity chromatography columns [13] in folded state were dialyzed against excess volumes of 1× transcription buffer. The purity of the recombinant proteins were >95% as determined by SDS/PAGE followed by coomassie blue staining. Recombinant proteins at optimal concentration, 0.4 µg per 15 µl of a newly prepared whole HeLa extract (~200 µg protein), were then added to the extracts for 30 min on ice before the start of the transcription reactions as specified above for each gene. Nuclear run-on transcription assay This assay was essentially performed as previously described by Prieto and McStay 2007 [43]. Nuclei were treated with DNase and Proteinase K and labeled RNA was isolated by phenol chloroform isoamylalcohol extraction and was then hybridized to probes immobilized on dot blots. The probes were pBluescript carrying human 5′ETS fragment of rDNA and 5S rRNA gene. Western blot analysis and immunoprecipitation assays Whole cell extracts or protein eluates from chromatography columns were separated in 12% polyacrylamide/0.1% SDS gels. Proteins were electrotransferred to nitrocellulose filters and immunoblotted with primary antibodies in 1 100–400 dilution. A 1 28,000 dilution of the corresponding secondary antibody was used. Blots were washed and bands were visualized using the ECL chemiluminescent kit, following instructions of the manufacturer (Amersham). RNase P from whole HeLa extracts was immunoprecipitated with polyclonal anti-Rpp antibodies or with monoclonal and polyclonal antibodies directed against Pol I, TAF I110, UBF, as described [10]. Immunoprecipitates were assayed for RNase P activity in processing of the 5′ leader sequence of a 32P-labeled precursor tRNA Tyr in 1× PA buffer mixed with 1× TNET buffer containing 2 mM Tris-HCl, pH 7.5, 35 mM NaCl, 0.1 mM Na 2EDTA, 1 mM 2-mercaptoethanol, 0.01% Triton X-100, 4 U of rRNasin and 12 µg Poly I C. Cleavage products were separated in 8% polyacrylamide/7 M urea gel. Chromatin immunoprecipitation analysis ChIP analysis was done essentially as previously described [10]. Asynchronized or synchronized HeLa cells were collected, washed with 1× PBS, and cross-linked with 0.5% NP-40/1× PBS containing 1% formaldehyde for 10 min at 37°C. Cells were rinsed with ice-cold 0.5% NP-40/1× PBS and incubated for 30 min in high-salt buffer (1 M NaCl, 0.5% NP-40, 1× PBS). Cells were collected, washed with 0.5% NP-40/1× PBS, and then resuspended in low-salt buffer (0.1 M NaCl, 0.1% NP-40, 10 mM Tris-HCl at pH 8.0, 1 mM EDTA). After 30 min, cells were centrifuged at 480×g for 10 min and subjected to 10 strokes through a 23-gauge needle. Nuclei obtained after centrifugation were resuspended in low-salt buffer containing 2% sarkosyl and transferred to a sucrose cushion (0.1 M sucrose, 0.1 M NaCl, 10 mM Tris-HCl at pH 8.0, 1 mM EDTA, 0.5% NP-40), and then spun for 10 min at 4000×g. The pellet was resuspended in TE (10 mM Tris-HCl at pH 8.0, 1 mM EDTA) and spun again, and genomic DNA was sheared by sonication (20×30 sec, duty cycle 80%) to produce stretches of chromatin of ~1000 base pairs (bp) in length. In the experiment described in , the genomic DNA was cut off to smaller pieces of ~500 bp. The sonicated material was diluted in 1/10 volume of ×11 NET buffer (1.65 M NaCl, 550 mM Tris-HCl at pH 7.4, 5.5 mM EDTA, 5.5% NP-40). Sonicated material (0.2 ml per each immunoprecipitation) was precleared for 30 min with 20 µl of Protein A/G Plus agarose beads (Santa Cruz Biotechnology) and then blocked with 2 µg of salmon sperm (Invitrogen). After centrifugation, the sample was subjected to IP overnight at 4°C using a nutating device with the appropriate antibody coupled to beads (coupling was for 6 h at 4°C) and in the presence of 4 µg of salmon sperm. Precipitated complexes were washed three times with 1 ml of RIPA buffer (50 mM Tris-HCl at pH 8.0, 150 mM NaCl, 0.1% SDS, 0.5% deoxycholate, 1% NP-40), three times with 1 ml of LiCl buffer (10 mM Tris-HCl at pH 8.0, 250 mM LiCl, 1 mM EDTA 0.5% deoxycholate, 0.5% NP-40), and three times with 1 ml of TE. Immunoprecipitated material was eluted twice with 200 µl of 1% SDS/TE and incubated overnight at 42°C in 0.4 ml volume of elution buffer containing 120 µg/ml Proteinase K. DNA was extracted twice with phenol/chloroform/isoamyl alcohol (25 24 1 v/v), ethanol-precipitated, washed with 70% ethanol, dried, and resuspended in 25 µL distilled water for use in PCR analysis. Each PCR reaction contained 2.5 µL of each primer (final concentration of 1 µM), 5 µl of 10× reaction mixture, and 4 µl of DNA for each IP sample. Input DNA was diluted in distilled water before PCR. PCR amplification programs and primer sequences for 5S rRNA, tRNA Tyr, U1 snRNA, ARPP P0 were described [10]. Primers for promoter rDNA were described in Philimonenko et al. 2004 [32]. Primers for 5.8S rDNA were 5′- CGACTCTTAGCGGTGGATCAC-3′ and 5′-AAGCGACGCTCAGACAGGCGT-3′, for 18S rDNA: 5′-CCTTTAACGAGGATCCATTGGA-3′ and 5′-GACACTCAGCTAAGA GCATCGAG -3′, for 28S rDNA: 5′-CTCTTCCTATCATTGTGAAGCAG-3′ and 5′-CAAATGTCTGAACCTGCGGTTC-3′ and for IGS: 5′-TGTTCTTGGGGGTGGGTTGAC-3′ and 5′-GAAGAGGTTCCGATGGGAAGTTG-3′. RNase H digestion assay Targeting the H1 RNA for specific cleavage by RNase H has been previously described [10]. Whole HeLa extract (15 µl; 10–15 mg/ml; not diluted) was incubated with 8 µg of H1-1 or scrambled H1-1sc deoxyoligonucleotide (Sigma, Israel) and 40 units of E. coli RNase H (Takara Bio, Inc.) for 45 min at 30°C in a final volume of 25 µl. The effect of this treatment on RNase P activity in the extracts was determined by examining 5′-end processing of 32P-labeled E. coli precursor tRNA Tyr and separation of the cleavage products in an 8% sequencing gel. 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