INTRODUCTION

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Transcription of a variety of cellular and viral genes is regulated, at least in part, at the level of elongation. Prior to the activation of these genes, RNA polymerase II (Pol II) initiates but pauses after synthesizing a short transcript. Transcription activation for these genes appears to be achieved by stimulating phosphorylation of the carboxy-terminal domain (CTD) of the largest subunit of Pol II (12, 31). The CTD, which consists of multiple repeats of the heptad sequence (YSPTSPS) ranging from 26 repeats in yeast to 52 in mammals, is essential in vivo, and transcription from many promoters is sensitive to CTD truncation (12, 18, 36, 55). Its role in elongation control has been further suggested by the observation that the CTD in paused Pol II is partially or unphosphorylated, but that in elongating Pol II is hyperphosphorylated (35).

One of the kinases that are thought to phosphorylate CTD in vivo for Pol II elongation is TFIIH, a complex of nine subunits (13, 43). TFIIH binds tightly to nonphosphorylated Pol II as a component of holoenzyme and dissociates from Pol II after transcription of 30 to 50 bp (53). It was suggested that the phosphorylation of CTD by TFIIH kinase may be important for promoter clearance for a certain promoter (2). However, recent studies indicated that the TFIIH-associated kinase is important to stimulate transcription elongation (1, 9, 10, 17, 38, 52). Although the exact role of TFIIH-mediated CTD phosphorylation in elongation control is not clear, strong evidence for the involvement of TFIIH in elongation comes from experiments that used antibodies against subunits of TFIIH in the Xenopus oocyte system. When anti-TFIIH antibodies against individual subunits were injected (52), transcription was inhibited due to the failure of elongation, suggesting an essential role of TFIIH in Pol II elongation.

TFIIH is present in the Pol II holoenzyme, where it is thought to regulate CTD phosphorylation at an early stage of transcription prior to the recruitment of other CTD kinases, such as P-TEFb, that are important for productive elongation (10, 56). Despite the presence of TFIIH, the CTD in Pol II holoenzyme remains unphosphorylated (28, 37). As the hyperphosphorylated Pol II (Pol IIO) cannot enter into a preinitiation complex (PIC), the unphosphorylated Pol II (Pol IIA) is thought to be the initiation-competent form in vivo (26, 27). One possibility is that a putative negative regulator that inhibits CTD phosphorylation may be present in the Pol II holoenzyme complex. In support of this view, previous in vitro transcription studies indicated that abortive elongation is an inherent property of the PICs derived from crude nuclear extract (24, 30, 49). In contrast, the reconstituted PICs with purified Pol II and general transcription factors can generate full-length transcripts efficiently, suggesting that elongation inhibitors may be associated with the Pol II holoenzyme.

The results in this study suggest that a ubiquitous nuclear protein, hnRNP U (heterogeneous nuclear ribonucleoprotein U) (14), may be one such elongation inhibitor in the Pol II holoenzyme. hnRNP U (120 kDa, 806 amino acids) is known as an RNA- and a scaffold/matrix attachment region DNA-binding protein. Approximately 50% of total hnRNP U is present in the nuclear matrix and 20% is tightly associated with chromatin, whereas half of hnRNP U in the remaining 30% soluble fraction is found in the hnRNP particles (16, 19, 20). hnRNP U is thought to participate in pre-mRNA processing together with other hnRNP proteins and/or to play a role in the higher-order organization of chromatin. Although most abundant proteins of the nuclear matrix are hnRNP proteins (33) and pre-mRNA is tightly associated with nuclear substructures, the RNA-binding RGG domain at its C terminus is dispensable for its interaction with nuclear matrix or chromatin. Rather, the N-terminal domain was found to be important for these interactions, while the RGG domain was shown to mediate interactions with other hnRNP proteins to form hnRNP particles.

This study suggests an unexpected function of hnRNP U as an inhibitor of Pol II elongation. We found that a fraction of hnRNP U is associated with the Pol II holoenzyme and is recruited to the promoter as part of a PIC. It appears that hnRNP U, via its middle domain, suppresses the CTD phosphorylation by TFIIH in the basal transcription machinery and inhibits Pol II elongation. This study suggests that the hnRNP U-mediated inhibition of TFIIH might be one of the mechanisms by which the CTD is maintained in an unphosphorylated state in the PIC. The involvement of hnRNP proteins in transcription regulation is not unprecedented. For example, hnRNP K has been shown to function as a transcription activator for c-myc gene expression (34, 48). The new role of hnRNP U as a Pol II elongation inhibitor underscores the diversity of potential roles of this class of proteins in a cell.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Plasmid construction. The cytomegalovirus (CMV) expression vectors for wild-type hnRNP U [HN(WT)] or various deletion mutants were constructed by inserting the full-length or corresponding deletion fragments in frame to a hemagglutinin epitope (HA) tag and the nuclear localization signal derived from simian virus 40 (SV40) T antigen. To construct various expression vectors in this study, the pCG-CMV expression vector was digested with XbaI and BamHI and ligated with a synthetic linker containing XbaI, KpnI, NotI, SalI, and BamHI sites. The nuclear localization signal derived from the SV40 T antigen was inserted into upstream NotI site (pCMV-1). For HA fusion proteins, the PCR fragment containing HA sequences was inserted into the XbaI/KpnI sites in pCMV-1 (pCMV-HA), and the corresponding hnRNP U fragments (NotI/SalI) were inserted in frame into pCMV-HA. All constructs made by PCR were verified by DNA sequencing.

Depletion of hnRNP U from HeLa nuclear extract. HeLa nuclear extract (5 to 7 µl or 60 µg of protein; typical amount used for one in vitro transcription reaction) was incubated with heparin-agarose (1/2 volume of the nuclear extract; Pharmacia) at 4°C for 1 h. The supernatant was collected and incubated with the hnRNP U antibody-protein A/G-Sepharose complex at 4°C for 1 h. For the anti-hnRNP U-Sepharose complex, 2 µl of the antibody was bound to 5 µl of protein A/G-Sepharose for 1 h at 4°C, and the immobilized beads were washed three times with buffer A (10 mM Tris, 10 mM NaCl, 5 mM MgCl₂, 1 mM dithiothreitol [DTT]) containing 0.5% Triton X-100. Although a considerable amount of hnRNP U was removed from nuclear extract after the heparin-agarose step, immunodepletion with anti-hnRNP U was necessary to remove the remaining hnRNP U protein.

Immunopurification of HA-HN from HeLa nuclear extract. HeLa cells (10⁷) were transfected with 20 µg of the expression vector for various HA-tagged hnRNP U (HA-HN) proteins. After 24 h of transfection, nuclear extract was prepared as described previously (22, 23). Prior to the addition of antibody, the nuclear extract was precleared by incubation with 1/10 volume of protein A/G-Sepharose. To 100 µl of nuclear extract containing 500 µg of protein, 15 µl of anti-HA antibody was added, and the mixture was incubated for 1 h at 4°C. The nuclear extract-antibody mixture was spun for 5 min at 14,000 rpm to remove any protein aggregates. Protein A/G-Sepharose was added (15 µl), and the mixture was incubated for 1 h at 4°C to precipitate the immune complexes. After five washes with buffer A, the bound protein was eluted with excess amount (5 µg) of HA peptide (Boehringer Mannheim).

Production of Strep-U in Schizosaccharomyces pombe. Modified S. pombe expression vector pESP-1-Strep (Stratagene), where the original glutathione-S-transferase (GST) tag was replaced with the streptococcal epitope (Strep) tag (WSHPQFEK), has been described elsewhere (4). After creation of NotI site in frame to the Strep tag, a NotI/BamHI fragment containing the hnRNP U cDNA was removed from the pCMV/hnRNP U construct and inserted into the NotI/BamHI-digested pESP-1-Strep vector. The transformed S. pombe cells were grown in the presence of 20 µM thiamine to repress the nmt1 promoter until expression was desired. Cells were lysed at 4°C in sorbitol buffer containing 0.5% Triton X-100 and protease inhibitors (Boehringer minitablets) by using a French press. Strep-U was purified by using a StrepTactin-Sepharose column (Genosys, The Woodlands, Tex.). Protein concentration was measured by the Bradford method (Bio-Rad).

In vitro transcription. For in vitro transcription reactions in Fig. 1 and 6, the linearized human immunodeficiency virus type 1 long terminal repeat (HIV-1 LTR)-chloramphenicol acetyltransferase (CAT) template (150 ng) was incubated for 30 min at 4°C with 60 µg of HeLa nuclear extract in a total volume of 25 µl containing transcription buffer (25 mM HEPES-KOH [pH 8.4], 7.5 mM MgCl₂, 4 mM DTT, 65 mM KCl, 10.5% glycerol). After PIC formation, a nucleoside triphosphate (NTP) mix (0.5 mM each ATP, GTP, CTP, and UTP) was added and the transcription reaction was performed for an additional 45 min at 30°C. RNA transcripts were purified and processed for hybridization in the presence of the probes. For Fig. 5B, a thymidine kinase (TK)-CAT expression plasmid (in which a point mutation was introduced to the EcoRI site in the CAT coding region to destroy the EcoRI site) was linearized with BamHI and biotinylated by using the Klenow enzyme in the presence of 10 µM biotin-16-dUTP (Boehringer Mannheim). To remove the biotin residue at the 3' end, the template was cleaved with SphI. PICs were formed by incubating the 5'-biotinylated template (500 ng) with 50 µg of HeLa nuclear extract in transcription buffer lacking NTPs for 30 min at 4°C. After formation of PICs, the templates were isolated by magnetic centrifugation following addition of streptavidin-coated magnetic beads (Promega) and washed with transcription buffer as described in a previous study (53). Prior to immunoprecipitation, the immobilized templates containing the PIC were digested with 100 U of EcoRI for 10 min at 30°C to cleave the TK promoter at position 80 and the magnetic beads were removed. After EcoRI digestion, the transcription complexes were immunoprecipitated and immunoblotted as indicated. For transcription initiation and elongation, PICs were formed as described above and subsequently incubated in the presence of various combinations of nucleotides such as ATP alone, ATP and CTP, or all four NTPs. Transcription reactions were performed for 20 min at 30°C. The templates were digested with EcoRI and washed in transcription buffer as described above, and the transcription complexes were immunoprecipitated. To monitor CTD phosphorylation in the reaction containing all four NTPs, the in vitro transcription reaction was carried out in the presence of 80 µCi of [-³²P]ATP.

RNase protection assay. The RNase T₁ protection assay was performed as described by the manufacturer (Ambion), using a range of input sample RNA amounts (1 to 20 µg) from transfected cells and a constant amount (5 × 10⁴ cpm) of probe. The linear increase in intensity of the protected fragment was seen with increasing amounts of RNA, indicating that the assay was performed under conditions of probe excess. Typically, 5 to 10 µg of RNA was hybridized to 5 × 10⁴ cpm of probe for the assay reported here. To generate the riboprobe, the linearized probe-containing plasmids were used as templates for T7 RNA polymerase.

Purification of Pol II holoenzyme. Pol II holoenzyme shown in Fig. 3A and B was purified as described previously (28). HeLa nuclear extract from 10⁹ cells was precleared with protein A/G-Sepharose and incubated with anti-Rap74 (250 µg; Santa Cruz)-bound protein A/G-Sepharose at 4°C overnight, washed extensively (50 mM HEPES-KOH [pH 7.8], 150 mM NaCl, 0.5% NP-40, 1% bovine serum albumin), and eluted with an excess amount of blocking peptide (Santa Cruz). The eluate containing approximately 100 µg of protein was fractionated on a gel filtration Sepharose CL-4B column (10-ml column, equilibrated in buffer containing 20 mM HEPES-KOH, 0.5 mM EDTA, 5 mM DTT, 0.01% NP-40, 0.1 mM phenylmethylsulfonyl fluoride, and 1 µg each of aprotinin, leupeptin, and pepstatin per ml). Since the peak of Pol II holoenzyme activity was shown to coelute with the peak of the blue dextran marker (2,000 kDa), fractions were collected as described previously (28). For immunoblotting, 30 µl of each 300-µl fraction was analyzed. All antibodies used for immunoblotting were purchased from Santa Cruz except the anti-CTD monoclonal antibody 8WG16 (QED Bioscience). Anti-hnRNP U and anti-hnRNP A1 were gifts from G. Dreyfuss and D. Levens. Holoenzyme immunopurification with anti-hnRNP U (Fig. 3C) was performed as follows. Per assay, 1 µl of monoclonal anti-hnRNP U was incubated with 20 µl of protein A/G-Sepharose beads for 4 h at 4°C. The beads were extensively washed in phosphate-buffered saline containing 0.1% Triton X-100 and incubated with nuclear extracts containing 200 µg of protein for 1 h at 4°C. The beads were then washed three times with 400 µl of washing buffer.

Immunoprecipitation with whole-cell lysate. Cells were solubilized with 0.5% Triton X-100 and 0.5% sodium deoxycholate (50 mM HEPES-KOH [pH 7.8], 0.1 M NaCl, 10 mM EDTA, 5 mM DTT, 0.1 mM phenylmethylsulfonyl fluoride, 20 µg of aprotinin per ml, 10 µg of leupeptin per ml) at 4°C for 15 min. The suspension was passed through a needle repetitively and centrifuged briefly. The supernatant was used for immunoblot and immunoprecipitation. For immunoprecipitation in Fig. 5A, a volume of lysate equivalent to 10⁶ cells was incubated with 2 µl of anti-hnRNP U antibody for 1 h at room temperature. After incubation with protein A/G-Sepharose beads for 2 h, the immunoprecipitates were washed and solubilized in sodium dodecyl sulfate (SDS) sample buffer. Pol II was detected by immunoblotting with anti-CTD monoclonal antibody 8WG.

TFIIH, Cdk8, and PITARLE (Cdk9) preparation. Anti-p62, anti-Cdk8, and anti-Cdk9 antibodies (2 µl of each; Santa Cruz) were incubated with HeLa nuclear extract (50 µg) precleared with protein-Sepharose beads for 1 h on ice. The complexes were immunoprecipitated by the addition of protein A/G-Sepharose beads and then washed five times in buffer containing 10 mM HEPES (pH 7.5), 10 mM KCl, 0.5 mM DTT, 0.5 mM EDTA, and 0.1% NP-40; the beads containing the immunoprecipitates (IPs) were used for the protein phosphorylation assay. Western blotting confirmed the presence of the specific kinase in each preparation (data not shown). To test whether other kinase activities are coimmunoprecipitated, the kinases were eluted from the beads and used for the CTD phosphorylation reaction in the presence or absence of the antibody specific to each kinase. CTD phosphorylation was blocked by the antibodies specific to these kinases (for TFIIH as shown in Fig. 4A; for Cdk8 and Cdk9, data not shown).

Protein phosphorylation assay. Protein phosphorylation assays were performed as described in other studies (10, 21, 32, 38). Briefly, reactions (20 µl) were performed in kinase buffer containing 50 mM Tris-HCl (pH 7.5), 5 mM MnCl₂, 40 mM KCl, 5 mM MgCl₂, 2% glycerol, 0.25 mg of bovine serum albumin per ml, 1 mM DTT, and 0.3% NP-40. Portions (from 5 µl of nuclear extract) of anti-p62 IP, anti-Cdk8 IP, or anti-Cdk9 IP beads were incubated with 20 µCi of [-³²P]ATP, 10 µM unlabeled ATP, 1 µg each of aprotinin, leupeptin, and pepstatin per ml, and a GST-CTD protein (200 ng) or recombinant TATA-binding protein (TBP; 200 ng; Promega) at 30°C for 1 h. The kinase reactions were terminated by addition of 5 µl of 5× SDS loading buffer. The GST-CTD fusion protein was expressed as described elsewhere (39). Various HA-HN proteins were immunopurified with anti-HA antibody 12C5A from HeLa nuclear extracts transfected with the expression vectors for various fusion proteins (20 µg of each CMV expression vector/10⁷ cells) and eluted from the beads in the presence of excess amount of HA peptide. A range of HA-HN proteins (5 to 50 ng, as judged by immunoblotting) was used for the CTD phosphorylation assay.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

hnRNP U as a potential basal repressor. Several studies have indicated that a number of nonhistone chromosomal- or nuclear matrix proteins that had been originally described as structural proteins have transcription activities. As a preliminary screen to identify repressors among this group of proteins, we measured CAT activities in cells transfected with expression vectors for several nuclear matrix proteins (hnRNP U, lamin B, topoisomerase II, hnRNP A1, and HMG I/Y) and found that hnRNP U inhibits the expression of the reporter genes tested (Table 1). Cotransfection of the expression vector for human hnRNP U with the reporter constructs containing Pol II promoters (HIV-1 LTR, TK, SV40, growth hormone [GH], and thyrotropin  [TSH]) resulted in repression in basal expression. The hnRNP U-mediated repression was released by the promoter-specific activators when tested in the previously described transcription activation system (22, 23) (Tat for the HIV-1 LTR promoter, Pit-1 for the GH promoter, Pit-1/AP-1 for the TSH promoter) (data not shown).

hnRNP U inhibits Pol II elongation. One reason for the repression shown in Table 1 might be the inhibition of basal transcription by hnRNP U. The effect of hnRNP U on transcription was examined in the HIV-1 LTR transcription system as a model. For the in vitro transcription assays shown in Fig. 1C, HeLa nuclear extract was depleted of hnRNP U, and the HA-HN immunopurified from transfected HeLa cells (HA-U in Fig. 1C) or the highly purified recombinant Strep-U (Fig. 1C) was added back. Conventional immunodepletion with anti-hnRNP U resulted in coimmunoprecipitation of Pol II holoenzyme (see Fig. 3C), and the depleted extract by this method did not support transcription efficiently (data not shown). For this reason, we depleted hnRNP U by incubating the nuclear extract with heparin-agarose prior to immunodepletion with anti-hnRNP U by taking advantage of the ability of hnRNP U to bind heparin with high affinity (40). Since heparin-agarose chromatography is frequently used in purification of transcription complexes, it was likely that the combination of heparin-agarose and anti-hnRNP U would successfully remove hnRNP U from nuclear extract without compromising the transcription activities of the nuclear extract. In Fig. 2B, immunoblot analysis indicated that hnRNP U was removed to levels below detection (lanes 1 to 3) following a combination treatment of heparin-agarose plus immunodepletion, whereas Pol II holoenzyme components such as Rpb-1 (Pol II), Rap74 (TFIIF), and p62 (TFIIH) or some other component of hnRNP particles such as hnRNP A1 (40) that had been reported to be resistant to the heparin treatment were retained in the nuclear extract (data not shown).

View larger version (28K): [in this window] [in a new window]   FIG. 1.   hnRNP U inhibits processive transcription by the HIV-1 LTR promoter. (A) RNA probes used for RNase protection assay. Probe A (200 nt) contains positions 100 to +80 of the HIV-1 LTR and the first 20 bp of the CAT gene. Probe B contains the C-terminal 200 nt of the CAT gene. (B) The presence of hnRNP U in nuclear extracts was examined by immunoblotting with anti-hnRNP U. hnRNP U depletion in lane 2 was done as described in Materials and Methods. In lane 3, Sp1 was depleted with anti-Sp1. C, control (undepleted extract). (C) In vitro transcription reactions were performed with the HIV-1 LTR (432 to +80) promoter and the hnRNP U-depleted HeLa nuclear extract (U-depleted) with or without addition of the HA-RN protein (HA-U; HeLa) or Strep-U (S. pombe) protein. Ct, control (undepleted extract). The recombinant Strep-U was purified to near homogeneity and detected by colloidal blue staining (Novex) on an SDS-6% polyacrylamide gel (Coomassie). In lanes 3 to 5 and 9 to 11, increasing amounts of the recombinant hnRNP U proteins containing 20, 40, and 100 ng of hnRNP U (as judged by immunoblotting with anti-hnRNP U [anti-U]) were added back. In lanes 6 and 12, the HA-U or Strep-U preparation containing 100 ng of hnRNP U (the same amount as used in lanes 5 and 11) was preincubated with anti-hnRNP U (2 µl) and added back. RNA transcripts were hybridized to antisense RNA probes A and B (A) and analyzed by RNase protection assays. The consistent level of the 60-nt transcripts served as an internal control. Negative controls from reactions performed without the template or without NTP, or performed in the presence of -amanitin or a nonspecific probe (128 to +100 region of the human TSH promoter), yielded no protected band (data not shown). For the RNase protection assays using probe A, relative levels of the long transcripts are indicated as percentages of the total protected RNA (short transcript + long transcript) (lanes 1 to 6, 4, 15, 12, 2, 0, 14; lanes 7 to 12, 3, 11, 5, 0.5, 0, and 13). (D) HeLa cells (5 × 106) were transfected with the HIV-1 LTR-CAT reporter (10 µg) and expression vector for hnRNP U as indicated. HN, hnRNP U. In lanes 4 and 5, the expression vector for Tat (pCMV/Tat) (20 ng) was cotransfected (Tat expression in transfected cells with only 20 ng of expression vector was not measurable by immunoblotting). Nuclear and cytoplasmic RNAs were extracted separately. The results shown were obtained by RNase protection assay using nuclear RNAs. Cytoplasmic RNAs showed identical results (data not shown). Pol III-driven transcripts from adenovirus VA1 (pSPVA1) were processed as previously described (52). A negative control containing RNA samples obtained from untransfected HeLa cells did not show any band (data not shown). Relative levels of the nonprocessive transcript as a percentage of the total protected RNA are indicated (lanes 1 to 5, 2.5, 0, 0, 26, and 24).

The hnRNP U-mediated block to elongation requires the CTD of Pol II. One of the mechanisms by which hnRNP U may block elongation is to inhibit CTD phosphorylation. Previous in vivo studies have shown that transcription from many promoters is sensitive to CTD truncation. However, transcription activation by Sp1 does not depend on CTD (54, 55), and transcription from an enhancerless promoter such as 4×Sp1-TK/CAT (hereafter called 4×Sp1) (Fig. 2A), consisting of a TATA box and four Sp1-binding sites, has been shown to be CTD independent in mammalian cells, including HeLa cells (7, 18). If hnRNP U blocks Pol II elongation by inhibiting CTD phosphorylation, CTD-independent transcription such as that from the 4×Sp1 promoter may not be affected by hnRNP U. To assess the CTD requirement in hnRNP U-mediated transcription repression, we used an approach developed by Gerber et al. (18), which relies on the efficient expression of -amanitin-resistant mutants of the large subunit of Pol II with different numbers of CTD repeats (Fig. 2A). -Amanitin treatment of cells transfected with these constructs results in inhibition of endogenous Pol II such that subsequent transcription depends on the exogenously expressed resistant mutant. To confirm that transcription from 4×Sp1 promoter in HeLa cells is CTD independent (7, 18), we transfected cells with the 4×Sp1 reporter and an expression vector for an -amanitin-resistant mutant with either 52 (wild-type; CTD-52) or 5 (CTD-5) repeats in the CTD (Fig. 2A). The effect of the residual endogenous Pol II activity that might have escaped -amanitin inhibition was assessed as described previously (7) by including a control transfection with the 4×Sp1 reporter and pUC19 without -amanitin-resistant mutants. Transcription from the 4×Sp1 promoter was not affected by CTD truncation, and the transcription signal was absent in control cells cotransfected with pUC19 (data not shown).

View larger version (21K): [in this window] [in a new window]   FIG. 2.   hnRNP U-mediated block of elongation requires the CTD of Pol II. (A) The 4×Sp1 reporter and expression vectors for -amanitin-resistant mutant of the Pol II largest subunit with CTD-52 and CTD-5. (B) Role of CTD in hnRNP U-mediated inhibition of Pol II elongation. HeLa cells (5 × 106 cells) were transfected with the 4×Sp1 or HIV-1 LTR reporter (10 µg) with or without expression vectors for hnRNP U (HN) (2 µg) and -amanitin-resistant mutants of Pol II (CTD-52 and CTD-5; 10 µg of each). -Amanitin (2.5 µg/ml) was added after 12 to 18 h of transfection, and the cells were incubated for an additional 48 h. Cytoplasmic RNAs were obtained and analyzed by RNase protection assay. For RNAs transcribed from the 4×Sp1 promoter (lanes 1 to 4), the 200-nt probe containing two Sp1 sites, TK promoter (37 to +55), and the first 45 bp of the CAT gene was used to protect the 100-nt fragment. The levels of exogenously expressed hnRNP U in the -amanitin-treated cells were monitored by measuring the transfected HA-HN level in cells treated in the same manner as those in lanes 3 and 4 (immunoblot HA-hn RNP U). For the RNase protection assays in lanes 5 to 8 probe A (Fig. 1A) was used. Relative levels of the nonprocessive transcripts as percentages of the total protected RNA are indicated (lanes 5 to 8, 6, 0, 2, and 1.5).

hnRNP U copurifies with Pol II holoenzyme in vivo. If hnRNP U functions as a transcription repressor, because hnRNP U is not a sequence-specific DNA-binding protein, it may be recruited to the promoter through protein-protein interactions. The possible association of hnRNP U with Pol II holoenzyme or TFIID in vivo has been examined. The Pol II holoenzyme was immunoprecipitated with anti-Rap74 (TFIIF) antibody (Fig. 3A) followed by gel filtration as described by Maldonado et al. (28) (Fig. 3B). Western blotting of the eluate from the anti-Rap74 IP (Fig. 3A) showed retention of Pol II (Rpb-1), hnRNP U, TFIIF (Rap74), TFIIH (Cdk7), and Cdk8. As previously reported (9, 28), however, transcription factors such as TBP, Sp1 (Fig. 3A), and TFIIB or Oct-1 (data not shown) were not detected. Similarly, unlike the eluates from the anti-Rap74 IP, those from the anti-Oct-1 IP (Fig. 3A) or anti-Sp1 IP (data not shown) did not contain hnRNP U, suggesting a possible interaction of hnRNP U with the Pol II holoenzyme complex. The addition of DNase or RNase to the immunoprecipitation reaction did not affect the results, indicating that hnRNP U was not artificially bound to the holoenzyme by contaminating DNA or RNA (data not shown). Fractionation of the anti-Rap74 IP eluate by gel filtration showed that hnRNP U copurifies with Pol II, TFIIF (Rap74), and TFIIH (Cdk7) with an apparent molecular mass of greater than 2 MDa (Fig. 3B). hnRNP A1, another component of hnRNP particles, was not detected, however. This rules out the possibility that the presence of hnRNP U in the fractions containing holoenzyme is due to the contaminating hnRNP particles comigrating with the holoenzyme.

View larger version (30K): [in this window] [in a new window]   FIG. 3.   hnRNP U is coimmunoprecipitated with Pol II holoenzyme. (A) The anti-Rap74 antibody immunoprecipitates hnRNP U from HeLa cell nuclear extract. Western blots of load (L), flowthrough (Fl), second wash (W), and eluate (E) with various antibodies are shown. The largest subunit of Pol II (Rpb-1) was detected with anti-CTD monoclonal antibody 8WG16 (QED Bioscience). (B) Fractionation of the anti-Rap74 IP eluate by gel filtration (Sepharose CL-4B) as described previously (28). (C) HeLa nuclear extract was immunoprecipitated with anti-hnRNP U. The eluate corresponding to 20 µl of nuclear extract was used for Western blotting. L, load; E, eluate. (D) The TFIID complex does not contain hnRNP U. L, load; E, eluate. (E) The Pol II-containing complex in panel C obtained by immunoprecipitation with anti-hnRNP U supports transcription in vitro. A linear G-free cassette template of the HIV-1 LTR (150 ng) was incubated with immobilized beads containing anti-hnRNP U immunoprecipitates (Pol II Holo) from 200 µg of HeLa nuclear extract and the mixture of ATP, CTP, and [-32P]UTP. The holoenzyme preparation was or was not supplemented with recombinant TBP (40 ng) and TFIIB (40 ng) (Promega) as indicated. The runoff transcript (390 nt) was resolved on a 6% polyacrylamide-urea gel.

hnRNP U inhibits the CTD phosphorylation by TFIIH in vitro. The results in Fig. 2 suggest that hnRNP U can inhibit CTD phosphorylation. One of the cellular targets of hnRNP U action could be a CTD kinase. Among a large number of kinases capable of phosphorylating the CTD in vitro, the targets for hnRNP U may be those in the PICs such as TFIIH-Cdk7 or -Cdk8 (13, 21, 25). Alternatively, hnRNP U may target CTD kinases that are not associated with the Pol II complex such as PITALRE (Cdk9), a catalytic subunit in the elongation factor P-TEFb complex that has been shown to play a role in productive elongation (29, 31, 56). These three CTD kinases have been widely postulated to play a role in CTD phosphorylation and elongation in vivo.

View larger version (37K): [in this window] [in a new window]   FIG. 4.   hnRNP U inhibits the CTD phosphorylation mediated by TFIIH-associated kinase. (A) hnRNP U inhibits kinase activities of TFIIH. For the TFIIH preparation in each lane in panel A, anti-p62 IP beads obtained by incubating 2 µl of anti-p62 antibody with 50 µg of HeLa nuclear extract were used. Lanes 1 to 3, TFIIH-mediated CTD phosphorylation. Lane 1, control (phosphorylation reaction was performed in kinase buffer in the presence of [-32P]ATP). Lane 2, CTD phosphorylation is blocked by anti-Cdk7 antibody. Lane 3, Cdk7 antigenic peptide releases the anti-Cdk7 blocking. Lanes 4 to 7, effect of HA-HN on TFIIH-mediated phosphorylation of GST-CTD (200 ng) or TBP (200 ng; Promega). HA-HN protein was immunopurified from transfected HeLa cells. HA-HN preparations containing 5, 25, and 50 ng of hnRNP U, as judged by immunoblotting with anti-hnRNP U, were used for lanes 4 to 7, respectively. Lane 8, HA-HN (50-ng equivalent [the same amount as used for lane 7]) was pretreated with anti-hnRNP U antibody (-HN; 2 µl) for 2 h at room temperature and added to the kinase reaction. Lanes 10 and 11, the amount of HA-HN was same as that used in lane 7 (50-ng equivalent). Lane 11, effect of GST-Tat (25 ng) on hnRNP U-mediated inhibition of CTD phosphorylation. (B) HA-HN does not inhibit Cdk8 and PITALRE (Cdk9) kinases. Cdk8 and PITARLE (Cdk9) were prepared essentially as described above for the TFIIH preparation. The amounts of HA-HN used in lanes 2 to 4 are same as those in lanes 5 to 7 in panel A. Lane 6, the amount of HA-HN that completely inhibited the TFIIH-mediated phosphorylation was used. HnRNP U did not inhibit the activities of Cdk8 or PITALRE (Cdk9) even when increasing amounts of substrate (HA-HN) or enzyme preparations (anti-Cdk8 IP and anti-Cdk9 IP) were tested (data not shown).

hnRNP U is recruited to the promoter in the form of a PIC and released from elongating Pol II. Pol II exists in two forms in cells, IIA and IIO. Although hnRNP U is a component of Pol II holoenzyme (Fig. 3) and the CTDs in the holoenzyme remain hypophosphorylated, these results do not necessarily indicate that hnRNP U is exclusively associated with Pol IIA in vivo. Because Pol IIO is predominantly associated with elongating complexes and discarded with chromatin during nuclear extract preparation (47), it was difficult to detect in nuclear extract. As reported in other studies (37), however, both IIO and IIA forms were detected in whole-cell lysate obtained from HeLa cells (Fig. 5A, lane 1). When the lysate was immunoprecipitated with anti-hnRNP U and immunoblotted with anti-CTD antibody (lane 2), only Pol IIA was detected, indicating that hnRNP U mainly associates with the nonprocessive form of Pol II in vivo.

View larger version (26K): [in this window] [in a new window]   FIG. 5.   HnRNP U is recruited to the promoter as a part of the PIC and dissociates from the elongating Pol II complex. (A) Anti-hnRNP U coimmunoprecipitates only the IIA form of Pol II in vivo. HeLa whole-cell lysate (lane 1) and the anti-hnRNP U immunoprecipitate (-HN IP; lane 2) were immunoblotted with anti-CTD antibody 8WG16. (B) The association of hnRNP U with Pol II was monitored in different stages of transcription in vitro as described in Materials and Methods. Transcription reactions were performed with the 5'-biotinylated TK-CAT template and HeLa nuclear extract. Transcription complexes formed from reactions incubated with DNA but without NTPs (lanes 1 and 2), with ATP (lanes 3 and 4), and with all four NTPs (lanes 5 and 6) were immunoprecipitated and immunoblotted as indicated. The hyperphosphorylation of Pol II in the reaction containing NTPs (lanes 5 and 6) was monitored in the presence of [32P]ATP.

The middle domain of hnRNP U is sufficient to mediate its Pol II holoenzyme association and its inhibition of the TFIIH kinase and Pol II elongation. HN(WT) has a modular structure, as indicated in Fig. 6A. The N-terminal domain (acidic and glutamine rich) is important for interaction with nuclear matrix and chromatin, while the RGG box-containing C-terminal domain is important for interaction with other hnRNP proteins to form hnRNP particles. To determine which domain of hnRNP U is essential for the different properties of hnRNP U described in this study (Pol II holoenzyme association, inhibition of the TFIIH kinase, and elongation), HA-HN proteins indicated in Fig. 6A were expressed in HeLa cells. In Fig. 6B (lanes 1 to 4 and 10 to 12), all HA-HN proteins were expressed efficiently in transfected cells. Pol II holoenzyme was isolated from cells expressing each HA-HN protein by immunoprecipitation with anti-Rap74. Because these preparations contain the mixture of Pol II holoenzyme complexes from transfected and untransfected cells, to enrich the population with the complexes containing HA-HN proteins, the anti-Rap 74 IPs were released and reprecipitated with anti-HA antibody. The Pol II holoenzymes released from the anti-HA IPs were tested for the presence of HA-HN proteins. As shown in Fig. 6B (lanes 5 to 8 and 14 to 16), only the HA-HN proteins containing the middle domain [HN(WT), HN/del N, HN/del C, and HN/Mid] were present in Pol II holoenzyme (lanes 5 to 8), indicating that the middle domain mediates the Pol II holoenzyme association. The presence of Rpb-1 (the largest subunit of Pol II) in the anti-Rap74 IPs was confirmed by immunoblotting with anti-CTD antibody (lower panels, lanes 5 to 8 and 14 to 16). As expected, in the final Pol II holoenzyme preparation released from the anti-HA IPs, Rpb-1 was present only in the complexes containing the HA-HN proteins with the middle domain (data not shown).

View larger version (30K): [in this window] [in a new window]   FIG. 6.   The middle domain of hnRNP U is sufficient to mediate its Pol II holoenzyme association and its inhibition of the TFIIH kinase and Pol II elongation. (A) HN(WT) and various HN deletion mutants. Modular structure of hnRNP U. Acidic (D, E), glutamine-rich (Q), and RNA-binding RGG domains are indicated. CMV expression vectors were constructed to contain the HA tag and the nuclear localization signal derived from the SV40 T antigen at the N termini of various hnRNP U fragments. (B) The middle domain of hnRNP U mediates its association with Pol II holoenzyme. HeLa cells (5 × 106 cells) were transfected with 10 µg of each expression vector. The nuclear extracts were immunoprecipitated with anti-Rap74. The bound proteins were released and reprecipitated with anti-HA antibody, and the resulting complexes were released in the presence of an excess amount of the HA peptide. Lanes 1 to 4 and 10 to 12, expression of each HA-HN protein in transfected cells probed with anti-HA; lanes 5 to 8 and 14 to 16, HA-HN proteins associated with the holoenzyme. In lanes 9 and 13, HA-HN(WT) was used as a marker. (C) The middle domain is essential for the inhibition of TFIIH-mediated CTD phosphorylation. Ct, control. Increasing amounts of each HA-HN protein were added to the kinase reaction as indicated. Similar amounts of each HA-HN protein measured by immunoblotting with anti-HA (data not shown) were used for comparison. (D) The middle domain of hnRNP U functions as a Pol II elongation block in vitro. In vitro transcription assays were performed as described for Fig. 1C. Similar amounts of HA-HN proteins as determined by immunoblotting (data not shown) were added back to the transcription reaction, and the transcripts were analyzed by RNase protection assay using probe A (Fig. 1A). Ct, control. (E) Exogenously expressed HA-HN proteins containing the middle domain inhibit elongation in vivo. HeLa cells were transfected with the HIV-1 LTR reporter (10 µg) and the expression vector for HA-HN proteins (2 µg) as described for Fig. 1D. RNase protection assays were performed with probe A (Fig. 1A), using the cytoplasmic RNAs.

hnRNP U can interact with TFIIH and inhibit CTD phosphorylation in vivo. Next, we tested if HA-HN could copurify with the endogeneous TFIIH in vivo. To obtain TFIIH that contains HA-HN proteins, the nuclear extract used in Fig. 6 was first immunoprecipitated with anti-p62, and the released TFIIH complexes from the anti-p62 IPs were reprecipitated with anti-HA. Immunoblotting of the resulting TFIIH complexes released from the anti-HA IP showed that HA-HN(WT) and only HA-HN proteins containing the middle domain were retained (lanes 1 to 8). These results indicated that hnRNP U may interact with TFIIH directly or indirectly in vivo and that the middle domain is sufficient to mediate this interaction (Fig. 7A). When the anti-p62 IP (first immunoprecipitation) was subjected to immunoblotting, p62, but not Rap74 or Rpb-1 (data not shown), was detected in all lanes, confirming that TFIIH, not Pol II holoenzyme, was immunoprecipitated. To confirm the specificity of the interaction of hnRNP U with TFIIH, we transfected HeLa cells with the expression vector for GAL4-Oct-1 (GAL4 DNA-binding protein fused to the N terminus of Oct-1) and immunopurified TFIIH from GAL4-Oct-1-containing nuclear extract in a manner similar to that described above. GAL4-Oct-1 was efficiently expressed in cells, as detected by immunoblotting with anti-GAL4 (lane 10), but was not detected in the TFIIH preparation after the first immunoprecipitation with anti-p62 (lane 9). As expected, GAL4-Oct-1 was not detected when the anti-p62 IP was reprecipitated with anti-GAL4 (data not shown). Consistently, the same pattern of binding was observed when the HA-HN proteins were incubated with the anti-p62-purified TFIIH complex in vitro (data not shown). To generate a stable TFIIH complex associated with hnRNP U in vivo, however, it may be necessary to use different purification schemes for the TFIIH complex such as biochemical purifications or immunopurification with antibodies against a different subunit of TFIIH. To date, hnRNP U has not been detected in the immunopurified TFIIH complex with anti-p62, possibly because of a transient interaction between hnRNP U and TFIIH in vivo.

View larger version (33K): [in this window] [in a new window]   FIG. 7.   HnRNP U can bind TFIIH and inhibit CTD phosphorylation in vivo. (A) Coimmunoprecipitation of endogenous TFIIH complex with HA-HN proteins (lanes 1 to 8). HeLa cells were transfected with 10 µg of each expression vector, and the nuclear extract was processed as described in the text. Retention of HA-HN proteins in the TFIIH complex was monitored by immunoblotting with anti-HA. For the negative control, cells were transfected with the vector for GAL4-Oct-1, and the TFIIH complex in the GAL4-Oct-1 containing nuclear extract was isolated as described above. GAL4-Oct-1 was abundantly expressed in cells, as detected by immunoblotting with anti-GAL4 (lane 10). In the TFIIH complex, however, GAL4-Oct-1 was not present (lane 9). (B) HnRNP U inhibits CTD phosphorylation in vivo. HeLa cells (5 × 106) were transiently transfected with 15 µg of expression vectors as indicated. Whole-cell lysates were subjected to SDS-polyacrylamide gel electrophoresis and immunoblotted with anti-CTD 8WG16. Ct, control (whole-cell lysate from untransfected cells).

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

This study reports a new role for hnRNP U: downregulation of CTD phosphorylation and inhibition of Pol II elongation. We showed that a fraction of hnRNP U is associated with the Pol II holoenzyme in vivo and is recruited to the promoter as part of a PIC. hnRNP U appears to dissociate from the Pol II complex at the early stage of transcription and is therefore absent from the elongating complex. The results shown in this study suggest that hnRNP U, as a component of the Pol II holoenzyme, may inhibit TFIIH-mediated CTD phosphorylation and repress Pol II elongation. Although tested with a limited number of promoters, these findings may have wider applications for Pol II transcription. Overall, this study identifies, for the first time, an elongation inhibitor associated with the Pol II holoenzyme.

HnRNP U confers a negative elongation potential to the basal transcription machinery. Although the role of the TFIIH-mediated CTD phosphorylation in transcription has not been clearly defined, previous studies suggest that it may be important for the processivity of Pol II. For example, the long-transcript production but not the short-transcript production from the HIV-1 LTR requires CTD and TFIIH (10). Together with the results of antibody injection experiments (52) where antibodies against each subunit of TFIIH injected into Xenopus oocytes selectively repressed long-transcript production from the coinjected HIV-1 LTR promoter, these findings indicated that long-transcript production from the HIV-1 LTR requires TFIIH. Considering that TFIIH is released from the transcription complex after a synthesis of short transcripts (53), it is difficult to explain how TFIIH selectively affects the long-transcript production that occurs after the release of TFIIH. One possibility is that the CTD phosphorylation by TFIIH does not have a major role in the early stage of transcription but is necessary to establish an elongation-competent form of Pol II (12). Indeed, several studies proposed that the early stage CTD phosphorylation by TFIIH is coupled to the functional transition to productive elongation (29, 56). In this manner, TFIIH would be able to influence the rate of processive elongation even after its release from the transcription complex, and accordingly, hnRNP U would be able to regulate Pol II processivity by regulating TFIIH. If hnRNP U controls Pol II elongation by inhibiting TFIIH kinase activities, it likely would not inhibit short-transcript production from the HIV-1 LTR or transcription from the 4×Sp1 promoter, that does not require CTD or the kinase activities of TFIIH.

The importance of TFIIH in Pol II elongation. It has been difficult to establish which of the CTD kinases plays a role in the in vivo phosphorylation of the CTD and subsequent elongation control, because doing so requires examination of Pol II phosphorylation in cells where specific CTD kinases have been inactivated. Given that CTD has multiple phosphorylation sites, the extent of phosphorylation may be differentially regulated according to the stages of transcription by distinct CTD kinases. Previous studies suggested that in vitro, TFIIH possesses a level of CTD kinase activities similar to that of P-TEFb, which is important for transition to productive elongation (32). Nevertheless, it is thought that the level of CTD phosphorylation by TFIIH in the Pol II complex in vivo is lower than that in the hyperphosphorylated CTD in the elongating complex (56). One of the functions of hnRNP U in the transcription machinery may be to control the level of CTD phosphorylation by modulating TFIIH kinase activities during the early stage of transcription. Such a tight regulation on TFIIH may be critical for productive elongation. If PITARLE (Cdk9), which is thought to be recruited to the promoter during the CTD hyperphosphorylation, is required for productive elongation as some studies have proposed (29, 31, 56), the results in this study suggest that TFIIH that phosphorylates the CTD before the recruitment of PITARLE (Cdk9) is necessary, although possibly not sufficient, for productive elongation. As has been proposed in other studies (29, 56), the TFIIH-mediated phosphorylation of CTD in the basal transcription machinery may be a prerequisite for the productive elongation that may involve other CTD kinases.

The hnRNP U-mediated block of elongation is a feature of higher organisms. To date, an hnRNP U homologue has not been found in yeast or Drosophila, which may reflect fundamental differences in transcription activation and elongation mechanisms between mammals and lower eukaryotes. For example, transcription activation in yeast is achieved primarily by acidic activators, whereas a variety of activators are used in higher organisms (45). Yeast Pol II cannot substitute human Pol II in the reconstituted in vitro transcription (8), although yeast and human TBPs are interchangeable (6). Further, Pol II from higher eukaryotes shows frequent pausing and arrest in the in vitro transcription analyses, while yeast Pol II in vitro exhibits close to in vivo elongation rates (30, 44, 46). Mammalian Pol II might use additional mechanisms to regulate CTD phosphorylation and elongation due to the additional number of repeats in its CTD. hnRNP U-mediated elongation control may represent one of those mechanisms.

Potential diverse biological roles of hnRNP U. The new role of hnRNP U described in this study, as a Pol II elongation inhibitor, underscores the diversity of roles that this class of proteins may play in a cell. Involvement of hnRNP proteins in transcription regulation is not unprecedented. Previous studies (34, 48) reported that hnRNP K is recruited to the c-myc gene promoter through a protein-protein interaction with TFIID to activate this gene (34). Further, a recent study reported that hnRNP U interacts with a ligand-dependent transcription factor, glucocorticoid receptor, suggesting a possible role of hnRNP U in the transcription of genes regulated by steroid hormones (15).