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Multiple interactions between RNA polymerase I, TIF‐IA and TAFI subunits regulate preinitiation complex assembly at the ribosomal gene promoter

Multiple interactions between RNA polymerase I, TIF‐IA and TAFI subunits regulate preinitiation... EMBO reports Multiple interactions between RNA polymerase I, TIF-IA and TAF subunits regulate preinitiation complex assembly at the ribosomal gene promoter 1 + Xuejun Yuan, Jian Zhao, Hanswalter Zentgraf , Urs Hoffmann-Rohrer & Ingrid Grummt Division of Molecular Biology of the Cell II and Applied Tumor Virology, German Cancer Research Center, D-69120 Heidelberg, Germany Received June 5, 2002; revised August 5, 2002; accepted September 2, 2002 In mammals, growth-dependent regulation of rRNA synthesis serum-starved or cycloheximide-treated cells lack this activity is brought about by the transcription initiation factor TIF-IA. and are therefore transcriptionally inactive. TIF-IA corresponds to TIF-IA is associated with a fraction of the TBP-containing factor factor C* (Brun et al., 1994) and yeast Rrn3p (Yamamoto et al., TIF-IB/SL1 and the initiation-competent form of RNA 1996). Both in yeast and mammals, Rrn3p/TIF-IA associates with polymerase I (Pol I). We investigated the mechanisms that a subpopulation of Pol I to form the transcriptionally active down-regulate cellular pre-rRNA synthesis and demonstrate enzyme, defined as the Pol I entity that is capable of initiating that nutrient starvation, density arrest and protein synthesis transcription from the rDNA promoter. inhibitors inactivate TIF-IA and impair the association of TIF-IA Besides being associated with Pol I, TIF-IA has been shown to with Pol I. Moreover, we used a panel of TIF-IA deletion interact with TIF-IB/SL1 (Miller et al., 2001). This suggests that, mutants to map the domains that mediate the interaction of by interacting with both Pol I and TIF-IB/SL1, TIF-IA targets tran- TIF-IA with Pol I and TIF-IB/SL1. We found that amino acids scriptionally active Pol I molecules to the rDNA promoter. 512–609 interact with two subunits of Pol I, RPA43 and PAF67, Given the essential role of TIF-IA in adapting rDNA transcription whereas a short, conserved motif (LARAK, amino acids to cell growth, we undertook a detailed analysis of the protein 411–415) is required for the association of TIF-IA with TAF 95 I domains that mediate the interaction of TIF-IA with Pol I and and TAF 68. The results uncover an interphase for essential I TIF-IB/SL1. We demonstrate that the C-terminal part of TIF-IA protein–protein interactions that facilitate Pol I preinitiation interacts with two subunits of Pol I, RPA43 and PAF67, whereas complex formation. an internal region of TIF-IA (LARAK, amino acids 411–415) mediates the interaction with TAF 68 and TAF 95. Importantly, I I INTRODUCTION the interaction between TIF-IA and Pol I is impaired in stationary, starved and cycloheximide-treated cells, under- Preinitiation complex formation at the mammalian ribosomal scoring the biological significance of these protein–protein RNA gene promoter is nucleated by the synergistic action of two interactions in initiation complex formation. DNA binding proteins, the HMG-box-containing upstream binding factor UBF (Jantzen et al., 1992) and the RNA RESULTS AND DISCUSSION polymerase I (Pol I)-specific TBP–TAF complex TIF-IB/SL1 (Comai et al., 1992; Eberhard et al., 1993). Pol I, together with two associated initiation factors, TIF-IA and TIF-IC, is recruited TIF-IA activity is down-regulated to the transcription start site by specific interaction with UBF and in growth-arrested cells TIF-IB/SL1 bound to the core element of the rDNA promoter (for a review, see Grummt, 1999). TIF-IA was initially characterized Fluctuations in cellular rRNA synthesis have been observed under a as an activity that complements inactive extracts from quiescent variety of conditions that affect cell growth and metabolism. mouse cells (Buttgereit et al., 1985). Extracts prepared from Previous studies revealed that the shutdown of rRNA synthesis Corresponding author. Molecular Biology of the Cell II, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany. Tel: +49 6221 423423; Fax: +49 6221 423404; E-mail: [email protected] 1082 EMBO reports vol. 3 | no. 11 | pp 1082–1087 | 2002 © 2002 European Molecular Biology Organization scientific report TIF-IA interactions Fig. 1. TIF-IA is inactivated in growth-arrested cells. (A) Northern blot. 45S pre-rRNA levels were monitored in 15 μg of RNA from exponentially growing, serum- starved, cycloheximide-treated and density-arrested cells. To normalize for variations of RNA loading, the blot was also hybridized with a probe complementary to cytochrome C oxidase (cox) mRNA. (B) Western blot. TIF-IA and Pol I from exponentially growing (lane 1), density-arrested (lane 2), amino-acid-starved (lane 3) or cycloheximide-treated (lane 4) FM3A cells were visualized on immunoblots with α-TIF-IA or α-RPA116 antibodies. (C) In vitro transcription. Nuclear extract proteins (50 μg) from growing (lane 1), density-arrested (lanes 2 and 5), cycloheximide-treated (lanes 3 and 6) and amino-acid-starved (lanes 4 and 7) FM3A cells were assayed for transcriptional activity in the absence (lanes 1–4) or presence (lanes 5–7) of 30 ng of recombinant TIF-IA. (D) TIF-IA is targeted by extracellular signals that inhibit growth. TIF-IA (30 and 60 ng) purified from exponentially growing (lanes 2 and 3), density-arrested (lanes 4 and 5), amino-acid- starved (lanes 6 and 7) and cycloheximide-treated (lanes 8 and 9) NIH 3T3 cells were assayed for their capability to restore the transcriptional activity of a nuclear extract from density-arrested FM3A cells (lane 1). (E) The interaction between TIF-IA and Pol I is impaired in growth-arrested cells. NIH 3T3 cells (1 × 10 ) were transfected with 2 μg of pcDNA3.1-Flag-hTIF-IA. To achieve confluency, 8 × 10 cells were seeded onto a 100 mm plate 12 h after transfection. Alternatively, cells were treated with cycloheximide or starved of cystine and methionine for 2 h before harvesting. Pol I was immunoprecipitated from cell lysates with α-RPA53 antibodies, and TIF-IA and Pol I were detected on western blots. during mitosis and early G phase is due to inactivation of TIF- treated or starved cells were virtually inactive (lanes 2–4). IB/SL1 and UBF (Heix et al., 1998; Klein and Grummt, 1999). Significantly, the addition of recombinant TIF-IA restored tran- Growth-dependent regulation of Pol I, on the other hand, has scriptional activity, reaching levels that are comparable to the been attributed to alterations in the amount or activity of TIF-IA control extract (lanes 5–7). This result demonstrates that TIF-IA, (Buttgereit et al., 1985; Schnapp et al., 1993). To study the but none of the other components of the Pol I transcription molecular mechanisms mediating growth-dependent regulation machinery, is targeted by diverse signaling pathways that of Pol I transcription, pre-rRNA levels of exponentially growing ultimately down-regulate the cell’s biosynthetic activity. NIH 3T3 cells were compared with those of serum-starved, If this conclusion is correct, then TIF-IA from growth-arrested cycloheximide-treated and density-arrested cells. Pre-rRNA cells should be transcriptionally inactive. To test this, TIF-IA was synthesis was monitored on northern blots using a labeled probe immunopurified from growing, confluent, starved and cyclohex- that hybridizes to the 5′ end of 45S pre-rRNA and specifically imide-treated NIH 3T3 cells, and equal amounts of TIF-IA were detects unprocessed pre-rRNA molecules. Figure 1A shows that assayed for their capability to restore Pol I transcription in pre-rRNA synthesis was markedly decreased in serum-starved, extracts from stationary cells. TIF-IA from growing cells activated cycloheximide-treated and density-arrested cells. This reduction Pol I transcription (Figure 1D, lanes 2 and 3). In contrast, TIF-IA of rRNA synthetic activity supports early studies demonstrating from density-arrested, starved and cycloheximide-treated cells that conditions that harm cellular metabolism impair Pol I tran- was transcriptionally inactive (lanes 4–9). Thus, the activity, but scription (Yu and Feigelson, 1972). not the amount, of TIF-IA is regulated by signals that impair cell To investigate whether the amount or activity of TIF-IA was metabolism and growth. altered in response to changes in cell growth, TIF-IA levels and TIF-IA has been shown to be part of the Pol I ‘holoenzyme’, transcriptional activity were compared in nuclear extracts from the enzyme moiety that is associated with most, if not all, factors density-arrested, amino-acid-starved or cycloheximide-treated required for transcription initiation (Saez-Vasquez and Pikaard, cells. All four extracts contained similar levels of TIF-IA and Pol I 1997; Seither et al., 1998; Albert et al., 1999). Moreover, TIF-IA (Figure 1B) but exhibited marked differences in transcriptional has been shown to interact with TIF-IB/SL1 (Miller et al., 2001), activity. Extracts from growing cells efficiently transcribed rDNA which suggests that TIF-IA bridges both protein complexes. To (Figure 1C, lane 1), whereas extracts from dense, cycloheximide- assess the function of TIF-IA in initiation complex formation, Pol I EMBO reports vol. 3 | no. 11 | 2002 1083 scientific report X. Yuan et al. Fig. 2. The C-terminus of TIF-IA interacts with RPA43 and PAF67. (A) Interaction of TIF-IA with Pol I and TIF-IB/SL1. Recombinant TIF-IA was incubated with 50 μl of partially purified cellular Pol I or TIF-IB/SL1 and incubated with control beads (lane 2) or bead-bound α-TIF-IA antibodies (lane 3). Associated Pol I and TIF-IB were visualized on western blots using α-RPA116 or α-TAF 95 antibodies. (B) GST pull-down assays. The indicated GST fusion proteins were immobilized on glutathione–Sepharose and incubated with S-labeled TIF-IA. TIF-IA bound to GST (lane 2), GST–RPA43 (lane 3), GST–RPA53 (lane 4) and GST–PAF67 (lane 5) was visualized by autoradiography. (C) RPA43 and PAF67 form a stable complex with TIF-IA. Extracts from E. coli expressing TIF-IA and GST (lane 1), TIF-IA and GST–RPA43 (lane 2), TIF-IA and GST–RPA53 (lane 3) or TIF-IA and GST–PAF67 (lane 4) were assayed on western blots for TIF-IA levels (lower panel). After incubation with glutathione–Sepharose, associated TIF-IA was monitored on western blots (upper panel). (D) Mutational analysis of TIF-IA/Pol I interactions. S-labeled TIF-IA and the respective mutants were incubated with immobilized GST (lanes 2 and 5), GST–RPA43 (lane 3) and GST–PAF67 (lane 6). TIF-IA was analyzed by SDS–PAGE and autoradiography. Twenty percent of the input proteins are shown in lanes 1 and 4. was precipitated from exponentially growing, confluent, starved 2000; Fath et al., 2001). Given that functionally important or cycloheximide-treated cells, and coprecipitated TIF-IA was protein–protein interactions are evolutionarily conserved, we detected on western blots. Consistent with TIF-IA being associ- examined whether the homologous mammalian subunit mediates ated with Pol I, significant amounts of TIF-IA were present in the the interaction with TIF-IA. To monitor interactions between TIF- immunoprecipitates from growing cells (Figure 1E, lane 5). IA and subunits of Pol I, pull-down experiments were performed Importantly, the amount of TIF-IA that was associated with Pol I (Figure 2B). S-labeled TIF-IA interacted with GST–mRPA43 but markedly decreased in growth-arrested cells (lanes 6–8). This result not with GST (lanes 2 and 3). No interaction was observed with is consistent with recent data in yeast and mammals demon- RPA53, the third largest subunit of Pol I (lane 4). Surprisingly, the strating dissociation of TIF-IA from Pol I in stationary or strongest binding was observed to PAF67 (lane 5), a 67 kDa cycloheximide-treated cells (Milkereit and Tschochner, 1998; protein that is tightly associated with the initiation-competent Cavanaugh et al., 2002). The current view is that inhibition of form of Pol I and was suggested to play an essential role in cell growth leads to hypophosphorylation and inactivation of recruiting Pol I to the rDNA promoter (Seither et al., 2001). TIF-IA and impairs the association of TIF-IA with Pol I. The interaction of TIF-IA with both PAF67 and RPA43 was substantiated by co-expressing TIF-IA in Escherichia coli TIF-IA interacts with two subunits of Pol I together with GST fusion proteins encoding PAF67, RPA53 or mRPA43 and capturing bound TIF-IA on glutathione–Sepharose. To study the interaction of TIF-IA with Pol I and TIF-IB/SL1, Flag- Consistent with the results above, TIF-IA was bound to GST–PAF67 tagged TIF-IA was incubated with cellular fractions containing and GST–RPA43 but not to control beads or GST–RPA53 partially purified Pol I or TIF-IB/SL1, precipitated with anti-FLAG (Figure 2C). Again, TIF-IA bound more efficiently to GST–PAF67 antibodies, and coprecipitated Pol I and TIF-IB/SL1 were analyzed on western blots. Consistent with TIF-IA mediating the than to GST–RPA43. Thus, TIF-IA interacts with two polypeptides of interaction between Pol I and TIF-IB/SL1, both Pol I and TIF-IB/SL1 Pol I, e.g. RPA43, a genuine subunit and PAF67, a Pol I-associated coprecipitated with TIF-IA (Figure 2A). factor that marks the initiation-competent enzyme moiety. This suggests that, by interacting with PAF67, TIF-IA may target a In Saccharomyces cerevisiae, Rrn3p has been found to interact with RPA43, a unique subunit of Pol I (Peyroche et al., functional subset of Pol I molecules into the initiation complex. 1084 EMBO reports vol. 3 | no. 11 | 2002 scientific report TIF-IA interactions Fig. 3. A conserved motif (LARAK) mediates the interaction of TIF-IA with TAF 95 and TAF 68. (A) Pull-down assay. Bead-bound Flag-tagged TIF-IA was I I incubated with extracts from Sf9 cells overexpressing HA-tagged TAF 48, TAF 68, TAF 95 or TBP, respectively. Proteins were separated by SDS–PAGE, and I I I TAF s were visualized on western blots using α-HA antibodies (lane 3). M2 beads saturated with the Flag epitope peptide were used as a control (lane 2). (B) The LARAK motif mediates the interaction of TIF-IA with TAF 68 and TAF 95. Flag-tagged TIF-IA (lane 3), TIF-IAΔ377–512 (lane 4), TIF-IAΔ95–163 (lane 5) and I I TIF-IA (lane 6) were incubated with HA-tagged TAF 68 or TAF 95. TIF-IA was immunoprecipitated, and associated TAF s were visualized on western blots. ΔLARAK I I I A scheme of the mutants and a sequence alignment of the LARAK domain of TIF-IA homologues are shown above. H.s., Homo sapiens; M.m., Mus musculus; S.p., Schizosaccharomyces pombe; S.c., Saccharomyces cerevisiae; C.e., Caenorhabditis elegans. (C) A synthetic peptide (LARAK) blocks the interaction between TIF-IA and TIF-IB/SL1. Immobilized TIF-IA was incubated at 4°C for 4 h with partially purified TIF-IB/SL1 in the absence (lane 3) or presence of 200 ng control peptide (EHLWKKLQDPSNPAI, lane 4) or LARAK peptide (YIGSFLARAKFITVKSC, lane 5) in 250 μl of buffer AM-100/0.5% NP-40. After stringent washing, bound TIF-IB/SL1 was analyzed on immunoblots with α-TAF 95 or α-TAF 68 antibodies. (D) The LARAK motif is essential for TIF-IA activity. In vitro I I transcription assays contained nuclear extracts from density-arrested FM3A cells and no exogenous TIF-IA (lane 1) or 15, 30 and 50 ng of wild-type TIF-IA (lanes 2–4) or TIF-IA (lanes 5–7). The amounts of recombinant TIF-IA added to the reactions was monitored on western blots with α-Flag antibodies (lower ΔLARAK panel). (E) Deletion or permutation of the LARAK motif abrogates TIF-IA activity in vivo. NIH 3T3 cells were cotransfected with 2.5 μg of the rDNA reporter plasmid and 1 or 2 μg of pBK-CMV-Flag-TIF-IA (lanes 2 and 3), pBK-CMV-Flag-TIF-IA (lanes 4 and 5) or pBK-CMV-Flag-TIF-IA (lanes 6 and 7). ΔLARAK LRAKA Transcripts from the rDNA reporter were monitored on northern blots. TIF-IA expression levels were monitored on western blots with α-Flag antibodies (lower panel). To map the respective domains of TIF-IA that interact with To define the domains of TIF-IA that mediate the interaction RPA43 and PAF67, GST pull-down experiments were performed with TAF s, wild-type and mutant TIF-IA were expressed in Sf9 with a set of TIF-IA deletion mutants. As shown in Figure 2D, cells and incubated with cell lysates containing HA-tagged mutants lacking the N-terminus (TIF-IA/ΔN94), a large internal TAF 95 or TAF 68, and bound TAF s were monitored on immuno- I I I region (TIF-IA/Δ377–512) or the C-terminal region (TIF-IA/ blots. Truncation of the N- or C-terminal part of TIF-IA did not ΔC609) efficiently associate with both RPA43 and PAF67. Deletion affect the interaction with either subunit (data not shown). Simi- of 95 amino acids from the C-terminus (TIF-IA/ΔC556) reduces larly, a mutant lacking amino acids 95–163 bound to both binding to PAF67 without affecting the interaction with RPA43. subunits with an efficiency comparable with wild-type TIF-IA Further truncation of the C-terminus (TIF-IA/ΔC499) abolishes (Figure 3B, lane 5). In contrast, deletion of residues 377–512 binding of TIF-IA to both RPA43 and PAF67. This suggests that abolished binding (lane 4). A search for conserved sequence two domains located between residues 512 and 609 mediate the elements within amino acids 377–512 revealed a motif (F/Y(L)ARAK) interaction between TIF-IA and Pol I. that is highly conserved among different species (Figure 3B). Deletion of the LARAK sequence (amino acids 411–415) abro- A conserved sequence element (LARAK) mediates gated TIF-IA binding to both TAF s (Figure 3B, lane 6), indicating the interaction between TIF-IA and TIF-IB that this motif serves an essential role in preinitiation complex assembly. To examine the interaction of TIF-IA with subunits of TIF-IB, Given that the LARAK motif mediates the interaction between immobilized TIF-IA was incubated with extracts from Sf9 cells TIF-IA and TIF-IB/SL1, a synthetic peptide harboring the LARAK that overexpress either of the four subunits of TIF-IB. Bound sequence should prevent the interaction of both factors. Indeed, proteins were analyzed on western blots using antibodies the LARAK peptide blocked the association of TIF-IA with TIF-IB/SL1 against TAF 95, TAF 68, TAF 48 and TBP, respectively. As I I I (Figure 3C, lane 5), whereas a control peptide had no effect (lane demonstrated in Figure 3A, TIF-IA interacted with TAF 95 and 4). Thus, amino acids 411–415 are required for the association of TAF 68, whereas no interaction of TIF-IA with TAF 48 or TBP was I I observed. TIF-IA with TIF-IB. EMBO reports vol. 3 | no. 11 | 2002 1085 scientific report X. Yuan et al. Consistent with the functional significance of the LARAK In vitro transcription assays. Standard reactions (25 μl) motif, TIF-IA was transcriptionally inactive. TIF-IA contained 30 ng of template pMr600/EcoRI, 12 mM Tris–HCl ΔLARAK ΔLARAK was incapable of complementing inactive extracts from density- pH7.9, 0.1 mM EDTA, 0.5 mM dithioerythritol, 5 mM MgCl , arrested cells in vitro (Figure 3D). Moreover, when assayed for 80 mM KCl, 12% glycerol, 0.66 mM each of ATP, CTP and GTP, activation of a Pol I reporter gene in vivo, wild-type but not 0.01mM UTP, 1 μCi [α- P]UTP and 50 μg of nuclear extract TIF-IA stimulated Pol I transcription (Figure 3E, lanes 1–5). proteins from FM3A cells. ΔLARAK Converting the LARAK sequence to LRAKA, a permutation that Immunoprecipitation and immunoblotting. Immunoprecipitations does not alter the charge of TIF-IA, also abolished TIF-IA activity were carried out using nuclear extracts from FM3A cells, lysates (lanes 6 and 7). These results demonstrate that the interaction from NIH 3T3 cells, or HEK 293T cells overexpressing Flag- between TIF-IA and TIF-IB is mediated by a discrete number of tagged TIF-IA. Cells were lysed in 1 ml of immunoprecipitation amino acids that have been evolutionary conserved. buffer (50 mM Tris-HCl pH 8.0, 2 mM EDTA, 150 mM NaCl, The finding that both TIF-IA and the yeast homologue Rrn3 is 0.5% Triton X-100, 1 mM DTT) and incubated for 4 h at 4°C with a key player in recruiting Pol I to the rDNA promoter under- the respective antibodies. After extensive washing, precipitated scores the essential role of this transcription factor in rRNA proteins were analyzed on immunoblots. synthesis. TIF-IA activity is regulated by diverse extracellular Protein–protein interaction experiments. Immobilized Flag- signals, indicating that this factor adapts Pol I transcription to tagged TIF-IA and control beads saturated with the Flag-epitope cell growth. TIF-IA is phosphorylated at multiple sites (unpub- peptide were incubated with lysates from Sf9 cells over- lished data), and data in yeast have revealed that reversible expressing recombinant subunits of TIF-IB. After incubation for phosphorylation of both Rrn3 and Pol I mediate formation and 2h at 4°C in buffer AM-100 plus 0.5% NP40 and washes, dissociation of the initiation complex (Fath et al., 2001). There- associated proteins were identified on western blots. To examine fore, elucidating the chains of events by which extracellular the association of TIF-IA with Pol I and TIF-IB, 500 ng of TIF-IA signals are transferred into the nucleolus and modify the activity expressed in Sf9 cells were incubated for 2 h at 4°C with 50 μl of of Rrn3/TIF-IA will unravel the mechanism of Pol I transcription partially purified Pol I or TIF-IB (H-400 or CM-400 fraction; initiation and the pathways that control this essential process. Schnapp and Grummt, 1996). After precipitation with anti-Flag antibodies, associated proteins were identified on western blots. To monitor the interaction between TIF-IA and subunits of Pol I METHODS in vitro, GST, GST–RPA43, GST–RPA53 and GST–PAF67 were Plasmids. The cDNA encoding TIF-IA (DDB/EMBL/GenBank attached to glutathione–Sepharose, and 10 μl of packed beads database accession No. AJ272050) was tagged with the Flag- containing 5 μg of GST or GST fusion proteins were incubated epitope and subcloned into various expression vectors. Mutant 35 with 7 μl of S-labeled TIF-IA in 100 μl of buffer (50 mM forms of TIF-IA were generated by in vitro mutagenesis using Tris–HCl pH 8.0, 0.5 mM EDTA, 150 mM NaCl, 0.5% Triton standard techniques. The rDNA reporter pMr1930-BH has been X-100, and protease inhibitors). After incubation for 1 h at room described previously (Budde and Grummt, 1999). The cDNA temperature, beads were washed, and bound proteins were encoding amino acids 1–248 of mouse RPA43 was amplified analyzed by SDS–PAGE and autoradiography. Alternatively, from plasmid IMAGp998M106538 (RZPD). TIF-IA and GST fusion proteins were co-expressed in E. coli Cell culture and transfection experiments. HEK 293T or NIH and bound to glutathione–Sepharose as described previously 3T3 cells were cultured in Dulbecco’s modified Eagle’s medium (Peyroche et al., 2000). supplemented with 10% FCS, 100 U/ml penicillin, 100 mg/ml streptomycin and 2 mM glutamine. Cells were harvested either ACKNOWLEDGEMENTS in the log-phase or after reaching confluence. Alternatively, cells were starved of cystine and methionine or treated with 0.1 mg/ml We thank Bettina Dörr for preparation and fractionation of cell cycloheximide for 2 h. For in vivo analysis of wild-type and extracts. We are grateful to all members of the laboratory for mutant TIF-IA, 5 × 10 cells were cotransfected with 2.5 μg of sharing reagents and advice. This work was supported by the reporter plasmid (pMr1930-BH) and different amounts of a German Cancer Research Center, the Deutsche Forschungsge- CMV-based expression vector encoding tagged wild-type or meinschaft and the Fonds der Chemischen Industrie. mutant forms of TIF-IA. 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(1998) A specialized form of RNA 69, 2833–2837. polymerase I, essential for initiation and growth-dependent regulation of rRNA synthesis, is disrupted during transcription. EMBO J., 17, 3692–703. DOI: 10.1093/embo-reports/kvf212 EMBO reports vol. 3 | no. 11 | 2002 1087 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png EMBO Reports Springer Journals

Multiple interactions between RNA polymerase I, TIF‐IA and TAFI subunits regulate preinitiation complex assembly at the ribosomal gene promoter

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EMBO reports Multiple interactions between RNA polymerase I, TIF-IA and TAF subunits regulate preinitiation complex assembly at the ribosomal gene promoter 1 + Xuejun Yuan, Jian Zhao, Hanswalter Zentgraf , Urs Hoffmann-Rohrer & Ingrid Grummt Division of Molecular Biology of the Cell II and Applied Tumor Virology, German Cancer Research Center, D-69120 Heidelberg, Germany Received June 5, 2002; revised August 5, 2002; accepted September 2, 2002 In mammals, growth-dependent regulation of rRNA synthesis serum-starved or cycloheximide-treated cells lack this activity is brought about by the transcription initiation factor TIF-IA. and are therefore transcriptionally inactive. TIF-IA corresponds to TIF-IA is associated with a fraction of the TBP-containing factor factor C* (Brun et al., 1994) and yeast Rrn3p (Yamamoto et al., TIF-IB/SL1 and the initiation-competent form of RNA 1996). Both in yeast and mammals, Rrn3p/TIF-IA associates with polymerase I (Pol I). We investigated the mechanisms that a subpopulation of Pol I to form the transcriptionally active down-regulate cellular pre-rRNA synthesis and demonstrate enzyme, defined as the Pol I entity that is capable of initiating that nutrient starvation, density arrest and protein synthesis transcription from the rDNA promoter. inhibitors inactivate TIF-IA and impair the association of TIF-IA Besides being associated with Pol I, TIF-IA has been shown to with Pol I. Moreover, we used a panel of TIF-IA deletion interact with TIF-IB/SL1 (Miller et al., 2001). This suggests that, mutants to map the domains that mediate the interaction of by interacting with both Pol I and TIF-IB/SL1, TIF-IA targets tran- TIF-IA with Pol I and TIF-IB/SL1. We found that amino acids scriptionally active Pol I molecules to the rDNA promoter. 512–609 interact with two subunits of Pol I, RPA43 and PAF67, Given the essential role of TIF-IA in adapting rDNA transcription whereas a short, conserved motif (LARAK, amino acids to cell growth, we undertook a detailed analysis of the protein 411–415) is required for the association of TIF-IA with TAF 95 I domains that mediate the interaction of TIF-IA with Pol I and and TAF 68. The results uncover an interphase for essential I TIF-IB/SL1. We demonstrate that the C-terminal part of TIF-IA protein–protein interactions that facilitate Pol I preinitiation interacts with two subunits of Pol I, RPA43 and PAF67, whereas complex formation. an internal region of TIF-IA (LARAK, amino acids 411–415) mediates the interaction with TAF 68 and TAF 95. Importantly, I I INTRODUCTION the interaction between TIF-IA and Pol I is impaired in stationary, starved and cycloheximide-treated cells, under- Preinitiation complex formation at the mammalian ribosomal scoring the biological significance of these protein–protein RNA gene promoter is nucleated by the synergistic action of two interactions in initiation complex formation. DNA binding proteins, the HMG-box-containing upstream binding factor UBF (Jantzen et al., 1992) and the RNA RESULTS AND DISCUSSION polymerase I (Pol I)-specific TBP–TAF complex TIF-IB/SL1 (Comai et al., 1992; Eberhard et al., 1993). Pol I, together with two associated initiation factors, TIF-IA and TIF-IC, is recruited TIF-IA activity is down-regulated to the transcription start site by specific interaction with UBF and in growth-arrested cells TIF-IB/SL1 bound to the core element of the rDNA promoter (for a review, see Grummt, 1999). TIF-IA was initially characterized Fluctuations in cellular rRNA synthesis have been observed under a as an activity that complements inactive extracts from quiescent variety of conditions that affect cell growth and metabolism. mouse cells (Buttgereit et al., 1985). Extracts prepared from Previous studies revealed that the shutdown of rRNA synthesis Corresponding author. Molecular Biology of the Cell II, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany. Tel: +49 6221 423423; Fax: +49 6221 423404; E-mail: [email protected] 1082 EMBO reports vol. 3 | no. 11 | pp 1082–1087 | 2002 © 2002 European Molecular Biology Organization scientific report TIF-IA interactions Fig. 1. TIF-IA is inactivated in growth-arrested cells. (A) Northern blot. 45S pre-rRNA levels were monitored in 15 μg of RNA from exponentially growing, serum- starved, cycloheximide-treated and density-arrested cells. To normalize for variations of RNA loading, the blot was also hybridized with a probe complementary to cytochrome C oxidase (cox) mRNA. (B) Western blot. TIF-IA and Pol I from exponentially growing (lane 1), density-arrested (lane 2), amino-acid-starved (lane 3) or cycloheximide-treated (lane 4) FM3A cells were visualized on immunoblots with α-TIF-IA or α-RPA116 antibodies. (C) In vitro transcription. Nuclear extract proteins (50 μg) from growing (lane 1), density-arrested (lanes 2 and 5), cycloheximide-treated (lanes 3 and 6) and amino-acid-starved (lanes 4 and 7) FM3A cells were assayed for transcriptional activity in the absence (lanes 1–4) or presence (lanes 5–7) of 30 ng of recombinant TIF-IA. (D) TIF-IA is targeted by extracellular signals that inhibit growth. TIF-IA (30 and 60 ng) purified from exponentially growing (lanes 2 and 3), density-arrested (lanes 4 and 5), amino-acid- starved (lanes 6 and 7) and cycloheximide-treated (lanes 8 and 9) NIH 3T3 cells were assayed for their capability to restore the transcriptional activity of a nuclear extract from density-arrested FM3A cells (lane 1). (E) The interaction between TIF-IA and Pol I is impaired in growth-arrested cells. NIH 3T3 cells (1 × 10 ) were transfected with 2 μg of pcDNA3.1-Flag-hTIF-IA. To achieve confluency, 8 × 10 cells were seeded onto a 100 mm plate 12 h after transfection. Alternatively, cells were treated with cycloheximide or starved of cystine and methionine for 2 h before harvesting. Pol I was immunoprecipitated from cell lysates with α-RPA53 antibodies, and TIF-IA and Pol I were detected on western blots. during mitosis and early G phase is due to inactivation of TIF- treated or starved cells were virtually inactive (lanes 2–4). IB/SL1 and UBF (Heix et al., 1998; Klein and Grummt, 1999). Significantly, the addition of recombinant TIF-IA restored tran- Growth-dependent regulation of Pol I, on the other hand, has scriptional activity, reaching levels that are comparable to the been attributed to alterations in the amount or activity of TIF-IA control extract (lanes 5–7). This result demonstrates that TIF-IA, (Buttgereit et al., 1985; Schnapp et al., 1993). To study the but none of the other components of the Pol I transcription molecular mechanisms mediating growth-dependent regulation machinery, is targeted by diverse signaling pathways that of Pol I transcription, pre-rRNA levels of exponentially growing ultimately down-regulate the cell’s biosynthetic activity. NIH 3T3 cells were compared with those of serum-starved, If this conclusion is correct, then TIF-IA from growth-arrested cycloheximide-treated and density-arrested cells. Pre-rRNA cells should be transcriptionally inactive. To test this, TIF-IA was synthesis was monitored on northern blots using a labeled probe immunopurified from growing, confluent, starved and cyclohex- that hybridizes to the 5′ end of 45S pre-rRNA and specifically imide-treated NIH 3T3 cells, and equal amounts of TIF-IA were detects unprocessed pre-rRNA molecules. Figure 1A shows that assayed for their capability to restore Pol I transcription in pre-rRNA synthesis was markedly decreased in serum-starved, extracts from stationary cells. TIF-IA from growing cells activated cycloheximide-treated and density-arrested cells. This reduction Pol I transcription (Figure 1D, lanes 2 and 3). In contrast, TIF-IA of rRNA synthetic activity supports early studies demonstrating from density-arrested, starved and cycloheximide-treated cells that conditions that harm cellular metabolism impair Pol I tran- was transcriptionally inactive (lanes 4–9). Thus, the activity, but scription (Yu and Feigelson, 1972). not the amount, of TIF-IA is regulated by signals that impair cell To investigate whether the amount or activity of TIF-IA was metabolism and growth. altered in response to changes in cell growth, TIF-IA levels and TIF-IA has been shown to be part of the Pol I ‘holoenzyme’, transcriptional activity were compared in nuclear extracts from the enzyme moiety that is associated with most, if not all, factors density-arrested, amino-acid-starved or cycloheximide-treated required for transcription initiation (Saez-Vasquez and Pikaard, cells. All four extracts contained similar levels of TIF-IA and Pol I 1997; Seither et al., 1998; Albert et al., 1999). Moreover, TIF-IA (Figure 1B) but exhibited marked differences in transcriptional has been shown to interact with TIF-IB/SL1 (Miller et al., 2001), activity. Extracts from growing cells efficiently transcribed rDNA which suggests that TIF-IA bridges both protein complexes. To (Figure 1C, lane 1), whereas extracts from dense, cycloheximide- assess the function of TIF-IA in initiation complex formation, Pol I EMBO reports vol. 3 | no. 11 | 2002 1083 scientific report X. Yuan et al. Fig. 2. The C-terminus of TIF-IA interacts with RPA43 and PAF67. (A) Interaction of TIF-IA with Pol I and TIF-IB/SL1. Recombinant TIF-IA was incubated with 50 μl of partially purified cellular Pol I or TIF-IB/SL1 and incubated with control beads (lane 2) or bead-bound α-TIF-IA antibodies (lane 3). Associated Pol I and TIF-IB were visualized on western blots using α-RPA116 or α-TAF 95 antibodies. (B) GST pull-down assays. The indicated GST fusion proteins were immobilized on glutathione–Sepharose and incubated with S-labeled TIF-IA. TIF-IA bound to GST (lane 2), GST–RPA43 (lane 3), GST–RPA53 (lane 4) and GST–PAF67 (lane 5) was visualized by autoradiography. (C) RPA43 and PAF67 form a stable complex with TIF-IA. Extracts from E. coli expressing TIF-IA and GST (lane 1), TIF-IA and GST–RPA43 (lane 2), TIF-IA and GST–RPA53 (lane 3) or TIF-IA and GST–PAF67 (lane 4) were assayed on western blots for TIF-IA levels (lower panel). After incubation with glutathione–Sepharose, associated TIF-IA was monitored on western blots (upper panel). (D) Mutational analysis of TIF-IA/Pol I interactions. S-labeled TIF-IA and the respective mutants were incubated with immobilized GST (lanes 2 and 5), GST–RPA43 (lane 3) and GST–PAF67 (lane 6). TIF-IA was analyzed by SDS–PAGE and autoradiography. Twenty percent of the input proteins are shown in lanes 1 and 4. was precipitated from exponentially growing, confluent, starved 2000; Fath et al., 2001). Given that functionally important or cycloheximide-treated cells, and coprecipitated TIF-IA was protein–protein interactions are evolutionarily conserved, we detected on western blots. Consistent with TIF-IA being associ- examined whether the homologous mammalian subunit mediates ated with Pol I, significant amounts of TIF-IA were present in the the interaction with TIF-IA. To monitor interactions between TIF- immunoprecipitates from growing cells (Figure 1E, lane 5). IA and subunits of Pol I, pull-down experiments were performed Importantly, the amount of TIF-IA that was associated with Pol I (Figure 2B). S-labeled TIF-IA interacted with GST–mRPA43 but markedly decreased in growth-arrested cells (lanes 6–8). This result not with GST (lanes 2 and 3). No interaction was observed with is consistent with recent data in yeast and mammals demon- RPA53, the third largest subunit of Pol I (lane 4). Surprisingly, the strating dissociation of TIF-IA from Pol I in stationary or strongest binding was observed to PAF67 (lane 5), a 67 kDa cycloheximide-treated cells (Milkereit and Tschochner, 1998; protein that is tightly associated with the initiation-competent Cavanaugh et al., 2002). The current view is that inhibition of form of Pol I and was suggested to play an essential role in cell growth leads to hypophosphorylation and inactivation of recruiting Pol I to the rDNA promoter (Seither et al., 2001). TIF-IA and impairs the association of TIF-IA with Pol I. The interaction of TIF-IA with both PAF67 and RPA43 was substantiated by co-expressing TIF-IA in Escherichia coli TIF-IA interacts with two subunits of Pol I together with GST fusion proteins encoding PAF67, RPA53 or mRPA43 and capturing bound TIF-IA on glutathione–Sepharose. To study the interaction of TIF-IA with Pol I and TIF-IB/SL1, Flag- Consistent with the results above, TIF-IA was bound to GST–PAF67 tagged TIF-IA was incubated with cellular fractions containing and GST–RPA43 but not to control beads or GST–RPA53 partially purified Pol I or TIF-IB/SL1, precipitated with anti-FLAG (Figure 2C). Again, TIF-IA bound more efficiently to GST–PAF67 antibodies, and coprecipitated Pol I and TIF-IB/SL1 were analyzed on western blots. Consistent with TIF-IA mediating the than to GST–RPA43. Thus, TIF-IA interacts with two polypeptides of interaction between Pol I and TIF-IB/SL1, both Pol I and TIF-IB/SL1 Pol I, e.g. RPA43, a genuine subunit and PAF67, a Pol I-associated coprecipitated with TIF-IA (Figure 2A). factor that marks the initiation-competent enzyme moiety. This suggests that, by interacting with PAF67, TIF-IA may target a In Saccharomyces cerevisiae, Rrn3p has been found to interact with RPA43, a unique subunit of Pol I (Peyroche et al., functional subset of Pol I molecules into the initiation complex. 1084 EMBO reports vol. 3 | no. 11 | 2002 scientific report TIF-IA interactions Fig. 3. A conserved motif (LARAK) mediates the interaction of TIF-IA with TAF 95 and TAF 68. (A) Pull-down assay. Bead-bound Flag-tagged TIF-IA was I I incubated with extracts from Sf9 cells overexpressing HA-tagged TAF 48, TAF 68, TAF 95 or TBP, respectively. Proteins were separated by SDS–PAGE, and I I I TAF s were visualized on western blots using α-HA antibodies (lane 3). M2 beads saturated with the Flag epitope peptide were used as a control (lane 2). (B) The LARAK motif mediates the interaction of TIF-IA with TAF 68 and TAF 95. Flag-tagged TIF-IA (lane 3), TIF-IAΔ377–512 (lane 4), TIF-IAΔ95–163 (lane 5) and I I TIF-IA (lane 6) were incubated with HA-tagged TAF 68 or TAF 95. TIF-IA was immunoprecipitated, and associated TAF s were visualized on western blots. ΔLARAK I I I A scheme of the mutants and a sequence alignment of the LARAK domain of TIF-IA homologues are shown above. H.s., Homo sapiens; M.m., Mus musculus; S.p., Schizosaccharomyces pombe; S.c., Saccharomyces cerevisiae; C.e., Caenorhabditis elegans. (C) A synthetic peptide (LARAK) blocks the interaction between TIF-IA and TIF-IB/SL1. Immobilized TIF-IA was incubated at 4°C for 4 h with partially purified TIF-IB/SL1 in the absence (lane 3) or presence of 200 ng control peptide (EHLWKKLQDPSNPAI, lane 4) or LARAK peptide (YIGSFLARAKFITVKSC, lane 5) in 250 μl of buffer AM-100/0.5% NP-40. After stringent washing, bound TIF-IB/SL1 was analyzed on immunoblots with α-TAF 95 or α-TAF 68 antibodies. (D) The LARAK motif is essential for TIF-IA activity. In vitro I I transcription assays contained nuclear extracts from density-arrested FM3A cells and no exogenous TIF-IA (lane 1) or 15, 30 and 50 ng of wild-type TIF-IA (lanes 2–4) or TIF-IA (lanes 5–7). The amounts of recombinant TIF-IA added to the reactions was monitored on western blots with α-Flag antibodies (lower ΔLARAK panel). (E) Deletion or permutation of the LARAK motif abrogates TIF-IA activity in vivo. NIH 3T3 cells were cotransfected with 2.5 μg of the rDNA reporter plasmid and 1 or 2 μg of pBK-CMV-Flag-TIF-IA (lanes 2 and 3), pBK-CMV-Flag-TIF-IA (lanes 4 and 5) or pBK-CMV-Flag-TIF-IA (lanes 6 and 7). ΔLARAK LRAKA Transcripts from the rDNA reporter were monitored on northern blots. TIF-IA expression levels were monitored on western blots with α-Flag antibodies (lower panel). To map the respective domains of TIF-IA that interact with To define the domains of TIF-IA that mediate the interaction RPA43 and PAF67, GST pull-down experiments were performed with TAF s, wild-type and mutant TIF-IA were expressed in Sf9 with a set of TIF-IA deletion mutants. As shown in Figure 2D, cells and incubated with cell lysates containing HA-tagged mutants lacking the N-terminus (TIF-IA/ΔN94), a large internal TAF 95 or TAF 68, and bound TAF s were monitored on immuno- I I I region (TIF-IA/Δ377–512) or the C-terminal region (TIF-IA/ blots. Truncation of the N- or C-terminal part of TIF-IA did not ΔC609) efficiently associate with both RPA43 and PAF67. Deletion affect the interaction with either subunit (data not shown). Simi- of 95 amino acids from the C-terminus (TIF-IA/ΔC556) reduces larly, a mutant lacking amino acids 95–163 bound to both binding to PAF67 without affecting the interaction with RPA43. subunits with an efficiency comparable with wild-type TIF-IA Further truncation of the C-terminus (TIF-IA/ΔC499) abolishes (Figure 3B, lane 5). In contrast, deletion of residues 377–512 binding of TIF-IA to both RPA43 and PAF67. This suggests that abolished binding (lane 4). A search for conserved sequence two domains located between residues 512 and 609 mediate the elements within amino acids 377–512 revealed a motif (F/Y(L)ARAK) interaction between TIF-IA and Pol I. that is highly conserved among different species (Figure 3B). Deletion of the LARAK sequence (amino acids 411–415) abro- A conserved sequence element (LARAK) mediates gated TIF-IA binding to both TAF s (Figure 3B, lane 6), indicating the interaction between TIF-IA and TIF-IB that this motif serves an essential role in preinitiation complex assembly. To examine the interaction of TIF-IA with subunits of TIF-IB, Given that the LARAK motif mediates the interaction between immobilized TIF-IA was incubated with extracts from Sf9 cells TIF-IA and TIF-IB/SL1, a synthetic peptide harboring the LARAK that overexpress either of the four subunits of TIF-IB. Bound sequence should prevent the interaction of both factors. Indeed, proteins were analyzed on western blots using antibodies the LARAK peptide blocked the association of TIF-IA with TIF-IB/SL1 against TAF 95, TAF 68, TAF 48 and TBP, respectively. As I I I (Figure 3C, lane 5), whereas a control peptide had no effect (lane demonstrated in Figure 3A, TIF-IA interacted with TAF 95 and 4). Thus, amino acids 411–415 are required for the association of TAF 68, whereas no interaction of TIF-IA with TAF 48 or TBP was I I observed. TIF-IA with TIF-IB. EMBO reports vol. 3 | no. 11 | 2002 1085 scientific report X. Yuan et al. Consistent with the functional significance of the LARAK In vitro transcription assays. Standard reactions (25 μl) motif, TIF-IA was transcriptionally inactive. TIF-IA contained 30 ng of template pMr600/EcoRI, 12 mM Tris–HCl ΔLARAK ΔLARAK was incapable of complementing inactive extracts from density- pH7.9, 0.1 mM EDTA, 0.5 mM dithioerythritol, 5 mM MgCl , arrested cells in vitro (Figure 3D). Moreover, when assayed for 80 mM KCl, 12% glycerol, 0.66 mM each of ATP, CTP and GTP, activation of a Pol I reporter gene in vivo, wild-type but not 0.01mM UTP, 1 μCi [α- P]UTP and 50 μg of nuclear extract TIF-IA stimulated Pol I transcription (Figure 3E, lanes 1–5). proteins from FM3A cells. ΔLARAK Converting the LARAK sequence to LRAKA, a permutation that Immunoprecipitation and immunoblotting. Immunoprecipitations does not alter the charge of TIF-IA, also abolished TIF-IA activity were carried out using nuclear extracts from FM3A cells, lysates (lanes 6 and 7). These results demonstrate that the interaction from NIH 3T3 cells, or HEK 293T cells overexpressing Flag- between TIF-IA and TIF-IB is mediated by a discrete number of tagged TIF-IA. Cells were lysed in 1 ml of immunoprecipitation amino acids that have been evolutionary conserved. buffer (50 mM Tris-HCl pH 8.0, 2 mM EDTA, 150 mM NaCl, The finding that both TIF-IA and the yeast homologue Rrn3 is 0.5% Triton X-100, 1 mM DTT) and incubated for 4 h at 4°C with a key player in recruiting Pol I to the rDNA promoter under- the respective antibodies. After extensive washing, precipitated scores the essential role of this transcription factor in rRNA proteins were analyzed on immunoblots. synthesis. TIF-IA activity is regulated by diverse extracellular Protein–protein interaction experiments. Immobilized Flag- signals, indicating that this factor adapts Pol I transcription to tagged TIF-IA and control beads saturated with the Flag-epitope cell growth. TIF-IA is phosphorylated at multiple sites (unpub- peptide were incubated with lysates from Sf9 cells over- lished data), and data in yeast have revealed that reversible expressing recombinant subunits of TIF-IB. After incubation for phosphorylation of both Rrn3 and Pol I mediate formation and 2h at 4°C in buffer AM-100 plus 0.5% NP40 and washes, dissociation of the initiation complex (Fath et al., 2001). There- associated proteins were identified on western blots. To examine fore, elucidating the chains of events by which extracellular the association of TIF-IA with Pol I and TIF-IB, 500 ng of TIF-IA signals are transferred into the nucleolus and modify the activity expressed in Sf9 cells were incubated for 2 h at 4°C with 50 μl of of Rrn3/TIF-IA will unravel the mechanism of Pol I transcription partially purified Pol I or TIF-IB (H-400 or CM-400 fraction; initiation and the pathways that control this essential process. Schnapp and Grummt, 1996). After precipitation with anti-Flag antibodies, associated proteins were identified on western blots. To monitor the interaction between TIF-IA and subunits of Pol I METHODS in vitro, GST, GST–RPA43, GST–RPA53 and GST–PAF67 were Plasmids. The cDNA encoding TIF-IA (DDB/EMBL/GenBank attached to glutathione–Sepharose, and 10 μl of packed beads database accession No. AJ272050) was tagged with the Flag- containing 5 μg of GST or GST fusion proteins were incubated epitope and subcloned into various expression vectors. Mutant 35 with 7 μl of S-labeled TIF-IA in 100 μl of buffer (50 mM forms of TIF-IA were generated by in vitro mutagenesis using Tris–HCl pH 8.0, 0.5 mM EDTA, 150 mM NaCl, 0.5% Triton standard techniques. The rDNA reporter pMr1930-BH has been X-100, and protease inhibitors). After incubation for 1 h at room described previously (Budde and Grummt, 1999). The cDNA temperature, beads were washed, and bound proteins were encoding amino acids 1–248 of mouse RPA43 was amplified analyzed by SDS–PAGE and autoradiography. Alternatively, from plasmid IMAGp998M106538 (RZPD). TIF-IA and GST fusion proteins were co-expressed in E. coli Cell culture and transfection experiments. HEK 293T or NIH and bound to glutathione–Sepharose as described previously 3T3 cells were cultured in Dulbecco’s modified Eagle’s medium (Peyroche et al., 2000). supplemented with 10% FCS, 100 U/ml penicillin, 100 mg/ml streptomycin and 2 mM glutamine. Cells were harvested either ACKNOWLEDGEMENTS in the log-phase or after reaching confluence. Alternatively, cells were starved of cystine and methionine or treated with 0.1 mg/ml We thank Bettina Dörr for preparation and fractionation of cell cycloheximide for 2 h. For in vivo analysis of wild-type and extracts. We are grateful to all members of the laboratory for mutant TIF-IA, 5 × 10 cells were cotransfected with 2.5 μg of sharing reagents and advice. This work was supported by the reporter plasmid (pMr1930-BH) and different amounts of a German Cancer Research Center, the Deutsche Forschungsge- CMV-based expression vector encoding tagged wild-type or meinschaft and the Fonds der Chemischen Industrie. mutant forms of TIF-IA. 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(1998) A specialized form of RNA 69, 2833–2837. polymerase I, essential for initiation and growth-dependent regulation of rRNA synthesis, is disrupted during transcription. EMBO J., 17, 3692–703. DOI: 10.1093/embo-reports/kvf212 EMBO reports vol. 3 | no. 11 | 2002 1087

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Published: Nov 1, 2002

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