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 ). 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 β [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
) (Tat for the HIV-1 LTR promoter, Pit-1 for the GH promoter, Pit-1/AP-1 for the TSH promoter) (data not shown).
TABLE 1 hnRNP U represses basal expression of the reportergenesa hnRNP U inhibits Pol II elongation.
One reason for the repression shown in Table 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. C, HeLa nuclear extract was depleted of hnRNP U, and the HA-HN immunopurified from transfected HeLa cells (HA-U in Fig. C) or the highly purified recombinant Strep-U (Fig. C) was added back. Conventional immunodepletion with anti-hnRNP U resulted in coimmunoprecipitation of Pol II holoenzyme (see Fig. C), 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. B, 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).
FIG. 2 hnRNP U-mediated block of elongation requires the CTD of Pol II. (A) The 4×Sp1 reporter and expression vectors for α-amanitin-resistant mutant of the Pol II largest subunit with CTD-52 and CTD-5. (B) Role of CTD in hnRNP U-mediated inhibition (more ...)
When the HIV-1 promoter is transcribed, two types of transcription complexes are observed: a processive type that results in full-length transcripts and the nonprocessive type that yields short transcripts consisting of the stable TAR RNA stem-loop (11
). In RNase protection assays with two antisense probes (Fig. A), transcription in the absence of Tat, using the undepleted HeLa nuclear extract (Fig. C, lanes 1 and 7), gave rise to short (~60-nucleotide [nt]) transcripts and elongated transcripts that protected a 100-nt fragment with probe A or a 200-nt fragment with probe B. The hnRNP U-depleted extract stimulated processive transcription (100 and 200 nt) but not nonprocessive transcription (60 nt) (Fig. C, lanes 2 and 8), while the extract depleted of hnRNP A1 by anti-hnRNP A1 (data not shown) contained the same levels of transcription as with the undepleted extract (lanes 1 and 7). Since it is not possible to produce a heparin-agarose-treated control extract without removing hnRNP U, it was not clear whether the observed stimulation in lanes 2 and 8 was due at least in part to the removal of an unknown inhibitor(s). However, add-back of increasing amounts of HA-HN (lanes 3 to 5) or Strep-U (lanes 9 to 11) to the hnRNP U-depleted extract resulted in repression of the processive transcription, whereas similar experiments with the HA-hnRNP A1 or Strep-hnRNP A1 did not show such an effect (data not shown). These results suggested that hnRNP U can repress Pol II elongation in vitro. In further support of this view, preincubation of the recombinant hnRNP U proteins with anti-hnRNP U (lanes 6 and 12) but not with control antibodies such as anti-hnRNP A1 or -Oct-1 (data not shown) abolished the repressive effect, indicating that hnRNP U, but not the contaminating proteins in the recombinant preparations, is responsible for the repressor activities.
To date, we have not been able to express a full-length recombinant hnRNP U in bacteria or in the in vitro translation system. Although obtained from two different sources, both HA-HN and Strep-U used for Fig. C contained hnRNP U, as confirmed by immunoblotting (data not shown), and their transcription activities were indistinguishable in our in vitro transcription assays. However, yeast cells expressing Strep-U were difficult to grow, and the yeast recombinant hnRNP U was very unstable. For these reasons, we chose to use the HA-HN proteins from HeLa cells for further assays.
A similar effect on Pol II processivity was seen in vivo (Fig. D). In the absence of Tat expression, overexpression of hnRNP U had no effect on nonprocessive transcription (60 nt) (Fig. D, lanes 1 to 3), but it inhibited processive transcription (100 and 200 nt) in a dose-dependent manner (lanes 1 to 3, panels I and II). Tat-activated processive transcription (100 nt; compare lanes 5 and 2) occurred in the presence of overexpressed hnRNP U. This release of the hnRNP U-mediated repression by Tat activation was consistently observed even in the presence of increasing amounts of transfected hnRNP U expression vector (up to 10 to 20 μg; data not shown). Because a direct interaction between Tat and hnRNP U was not observed in the in vitro GST pull-down assay (21a
), it is unlikely that the effect of Tat shown in lane 5 resulted from blocking or titrating hnRNP U with Tat. The hnRNP U effect was specific for Pol II, since Pol III-dependent VA1 transcription was not affected (Fig. D, lower panels).
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
), and transcription from an enhancerless promoter such as 4×Sp1-TK/CAT (hereafter called 4×Sp1) (Fig. A), consisting of a TATA box and four Sp1-binding sites, has been shown to be CTD independent in mammalian cells, including HeLa cells (7
). 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 α-amanitin-resistant mutants of the large subunit of Pol II with different numbers of CTD repeats (Fig. A). α-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
), we transfected cells with the 4×Sp1 reporter and an expression vector for an α-amanitin-resistant mutant with either 52 (wild-type; CTD-52) or 5 (CTD-5) repeats in the CTD (Fig. A). The effect of the residual endogenous Pol II activity that might have escaped α-amanitin inhibition was assessed as described previously (7
) by including a control transfection with the 4×Sp1 reporter and pUC19 without α-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).
We then tested whether hnRNP U overexpression would affect transcription from the 4×Sp1 promoter. Results of RNase protection assays indicate that hnRNP U does not inhibit transcription from this promoter (Fig. B, lanes 1 and 2). Similar results were obtained for cells transfected with increasing amounts of hnRNP U expression vector (up to 10 μg), suggesting that this resistance is not due to an insufficient amount of exogenously expressed hnRNP U (data not shown). CTD truncation and the overexpression of hnRNP U also did not affect transcription from the 4×Sp1 promoter when cells were treated with α-amanitin following transfection with the reporter and expression vectors for Pol II mutants and hnRNP U (lanes 3 and 4). To rule out the possibility that the absence of repression in elongation in the CTD-5-expressing cells is due to an insufficient amount of the exogenously expressed hnRNP U, the levels of hnRNP U expression were monitored in cells transfected with the expression vector for HA-HN (pCMV/HA-hnRNP U) and treated in the same manner as those in lanes 3 and 4. The results of immunoblotting with anti-HA (Fig. B) indicated that the CTD-52- and CTD-5-expressing cells expressed similar levels of HA-HN, in agreement with a previous report that transcription from the CMV promoter is not sensitive to CTD truncation (5
). Overall, the results in Fig. B suggest that hnRNP U does not inhibit CTD-independent transcription from the 4×Sp1 promoter.
The CTD requirement in hnRNP U-mediated repression was further examined in cells transfected with the HIV-1 LTR reporter and the α-amanitin-resistant mutants of Pol II (Fig. B, lanes 5 to 8). As shown in other studies (7
), the level of processive transcription (100 nt), but not nonprocessive transcription (60 nt), was reduced upon CTD truncation (compare lanes 5 and 7). When hnRNP U was overexpressed in the presence of the wild-type CTD-52 (lane 6), only processive transcription (100 nt) was inhibited (compare lanes 5 and 6), consistent with the results in Fig. D. When the CTD-5 construct was used (lane 8), however, the low level of processive transcription (100 nt) was resistant to hnRNP U as was nonprocessive transcription (60 nt) (compare lanes 7 and 8), indicating that CTD-dependent transcription of the HIV-1 LTR is sensitive to hnRNP U. The simplest hypothesis suggested by the results in Fig. is that the hnRNP U-mediated block to elongation may require the CTD of Pol II.
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. A) followed by gel filtration as described by Maldonado et al. (28
) (Fig. B). Western blotting of the eluate from the anti-Rap74 IP (Fig. A) showed retention of Pol II (Rpb-1), hnRNP U, TFIIF (Rap74), TFIIH (Cdk7), and Cdk8. As previously reported (9
), however, transcription factors such as TBP, Sp1 (Fig. A), 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. A) 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. B). 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.
The possible association of hnRNP U with Pol II holoenzyme was further suggested by the observation that eluates from the anti-hnRNP U IP contained Pol II, TFIIF, Cdk7, and hnRNP U but not TBP (Fig. C). In contrast, the TFIID complex immunoprecipitated with anti-TBP did not contain hnRNP U but did contain known components of TFIID such as TAF-250, TAF-130, and TBP (Fig. D). To determine if the Pol II-containing complex immunopurified with anti-hnRNP U in Fig. C was competent for transcription, the anti-hnRNP U IP immobilized on washed beads was added to the transcription reaction (Fig. E). Specific transcription depended on the addition of recombinant TBP and TFIIB (lane 4) and was sensitive to α-amanitin (data not shown). In contrast, no transcription was observed with the IPs generated by anti-hnRNP A1 (data not shown). These results suggest that hnRNP U may be recruited to promoters through its association with Pol II holoenzyme. How hnRNP U is incorporated into holoenzyme and what fraction of holoenzyme is associated with hnRNP U in vivo remain unknown.
hnRNP U inhibits the CTD phosphorylation by TFIIH in vitro.
The results in Fig. 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
). 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
). These three CTD kinases have been widely postulated to play a role in CTD phosphorylation and elongation in vivo.
The effect of hnRNP U on CTD phosphorylation by these kinases was assayed on GST-CTD by using HA-HN (Fig. ). The immunopurified kinase preparations used in these experiments did not contain any contaminating hnRNP U (data not shown). All three kinases phosphorylated GST-CTD (Fig. A, lanes 1 and 4, and B, lanes 1 and 5), as reported in other studies (10
). This phosphorylation was blocked when each kinase was pretreated with the corresponding antibody and the antibody-specific blocking was released in the presence of the antigenic peptide (for TFIIH, Fig. A, lanes 2 and 3; for other kinases, data not shown), indicating that contaminating kinases were not responsible for the phosphorylation shown. When HA-HN was added to the TFIIH-mediated phosphorylation reaction, 32
P incorporation into both hypo- and hyperphosphorylated forms (GST/CTD-A and GST/CTD-O) was inhibited in a dose-dependent manner (lanes 4 to 7). As with CTD, hnRNP U inhibited phosphorylation of TBP, another substrate of TFIIH (lower panel, lanes 4 to 7). Preincubation of the immunopurified HA-hnRNP U with anti-hnRNP U (lane 8) but not with control antibodies such as anti-hnRNP A1 and anti-Oct-1 (data not shown) effectively neutralized the inhibition, indicating that contaminating kinase inhibitor activities or phosphatases in the HA-HN preparation are not responsible for the observed inhibition. Furthermore, if HA-HN was added to the reaction 1 h after the start of the kinase reaction, TFIIH-mediated phosphorylation was not inhibited (data not shown). In contrast to the effect on TFIIH, Cdk8 and PITARLE (Cdk9) activities (Fig. B) were not affected by the amount of HA-HN that completely inhibited the TFIIH-mediated reaction. These results indicate that hnRNP U specifically inhibits TFIIH-associated kinase and that this inhibition is not due to phosphatase activities. We then tested whether a transactivator such as Tat that has been reported to bind the Cdk7 subunit of TFIIH to activate TFIIH (10
) might be able to release the inhibitory effect of hnRNP U. Interestingly, the inhibitory effect of hnRNP U on the TFIIH-mediated phosphorylation in vitro was neutralized by Tat (Fig. A, lanes 9 to 11). Further biochemical studies are required to elucidate the mechanism for this neutralization by Tat whether Tat might change conformation of TFIIH or compete with hnRNP U for binding to the TFIIH complex.
The hnRNP U-mediated inhibition in CTD phosphorylation in Fig. A can be attributed to many different reasons. One possibility is that hnRNP U, by binding to the CTD, sterically hinders its phosphorylation. However, a direct interaction between CTD and hnRNP U was not detected in GST pull-down assays (data not shown). Further, the result in Fig. B that hnRNP U did not inhibit the Cdk8- or PITARLE (Cdk9)-mediated CTD phosphorylation makes this possibility unlikely. This result also rules out the possibility that hnRNP U inhibits the kinase reaction by binding ATP nonspecifically. The second possibility is that hnRNP U competes with CTD as a substrate for TFIIH kinase. This is unlikely because 32P incorporation into hnRNP U (>120 kDa) was not detected in the in vitro phosphorylation reaction (Fig. A). The third possibility is that hnRNP U disrupts the assembly of TFIIH. If this is the case, however, it is unlikely that Tat would neutralize the effect of hnRNP U (Fig. A, lanes 9 to 11). Moreover, the observation that TBP phosphorylation by TFIIH was also inhibited by hnRNP U (Fig. A, lanes 4 to 7) suggests that hnRNP U inhibits TFIIH activity rather than sterically hindering CTD phosphorylation sites on TFIIH. The fourth possibility is that hnRNP U interacts with TFIIH (see Fig. ) and possesses TFIIH-specific kinase inhibitor activities.
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 (more ...) 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. ) 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. A, 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.
If Pol IIO is derived from Pol IIA as currently thought, these results imply that hnRNP U dissociates from Pol IIA during or after CTD phosphorylation. To test this hypothesis, the association of hnRNP U with Pol II was monitored during different stages of transcription in vitro on the immobilized TK (−105 to +55) template. The HIV-1 LTR template was not used, because HIV-1 transcription results in a mixture of paused (IIA) and elongating (IIO) complexes that are difficult to isolate separately. A PIC was formed on the 5′-biotinylated TK template with HeLa nuclear extract (without NTP), and the template was immobilized by binding to streptavidin-coated magnetic beads. The DNA templates containing the transcription complexes were released from magnetic beads by restriction enzyme digestion, and the resulting transcription complexes were immunoprecipitated with anti-Rap74 or anti-TBP antibody (Fig. B, lanes 1 and 2). Components of the holoenzyme and TFIID including TFIIH (Cdk7), TFIIF (Rap74), TBP, and hnRNP U were present in both IPs, indicating that hnRNP U is recruited to the promoter with holoenzyme (Fig. ) and incorporated into the PIC. When the transcription reaction was performed with ATP alone (lanes 3 and 4) or with ATP and CTP, which would allow formation of the first phosphodiester bond from the TK promoter (data not shown), hnRNP U and Cdk7 were still present in the IPs with anti-Rap74 and anti-TBP antibodies. When NTP was added to allow transcription elongation (Fig. B, lanes 5 and 6), the anti-TBP antibody could no longer coimmunoprecipitate Pol II with TBP (TFIID) and the anti-Rap74 could not immunoprecipitate TBP (TFIID) with Pol II, indicating that Pol II had left the initiation complex. When the hyperphosphorylation of Pol II was monitored with [γ-32P]ATP in the reaction containing all NTPs, the anti-Rap74 IP (lane 5, bottom) but not the anti-TBP IP (lane 6, bottom) contained Pol IIO, further confirming that Pol II was released from the initiation complex. At this stage of transcription, hnRNP U and Cdk7 were not detected in the anti-TBP- and anti-Rap74 IPs (lanes 5 and 6). These results suggest that hnRNP U and TFIIH dissociate from the initiation complex prior to productive elongation and that the release of hnRNP U and TFIIH requires transcription.
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. A. 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. A were expressed in HeLa cells. In Fig. B (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. B (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).
We then tested which domain is important for the hnRNP U-mediated inhibition of TFIIH kinase activity. When the immunopurified HA-HN proteins were added to the TFIIH kinase reaction as described in Fig. , hnRNP U with the middle domain deletion (HN/del Mid) and the truncated protein containing the N or C terminus only (HN/N or HN/C) showed no inhibition (Fig. C, lanes 2 to 4), whereas the middle domain-containing mutants (HN/Mid, HN/del N, and HN/del C) all showed inhibition (lanes 5 to 7), indicating that the middle domain inhibits the TFIIH kinase.
The middle domain was also sufficient for the inhibition of Pol II elongation from the HIV-1 LTR in vitro (Fig. D) and in vivo (Fig. E). When various HA-HN proteins were added back to the in vitro transcription reaction using the HeLa nuclear extract depleted of hnRNP U (Fig. D), the middle domain-containing mutants (lanes 7 to 9), but not the mutants lacking the middle domain (lanes 4 to 6), restored elongation inhibition. Inhibition of elongation was also dependent on the middle domain in cells transfected with the expression vectors for the various HA-HN proteins (Fig. E).
These results indicated that the middle domain of hnRNP U is sufficient for interaction with Pol II holoenzyme and for inhibition of TFIIH kinase and Pol II elongation, a function that has not been described previously for any protein.
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. 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. A). 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.
To test whether the inhibition of TFIIH kinase activity by hnRNP U and the ability of hnRNP U to associate with Pol II holoenzyme and TFIIH are correlated to hypophosphorylation of the CTD in vivo, HeLa cells were transfected with expression vectors for HA-HN(WT) and HA-HN/Mid, and the levels of Pol IIA and Pol IIO in whole-cell lysates were compared with those in nontransfected cells by immunoblotting (Fig. B). As expected, the Pol IIO form was substantially decreased in lysates from cells expressing HA-HN proteins, which can inhibit TFIIH kinase activity and associate with Pol II, HA-HN(WT), and HA-HN/Mid (lanes 2 and 3). In contrast, cells expressing HA-HN/N, HA-HN/C, or Sp1 (lanes 4 to 8) showed no difference from the control (lanes 5 and 8).
Overall, this study shows correlative evidence linking hnRNP U-mediated inhibition in CTD phosphorylation by TFIIH to the hnRNP U-mediated repression in Pol II elongation. Together, these results suggest that a subfraction of hnRNP U is recruited to the Pol II holoenzyme, where it appears to inhibit CTD phosphorylation by downregulating TFIIH and may thereby repress Pol II elongation. Although it remains unknown how hnRNP U might regulate TFIIH, our preliminary results suggest that hnRNP U specifically interacts with Cdk7 but not with other subunits of TFIIH in vitro (21a
). Detailed mutational analyses of the middle domain of hnRNP U and the Cdk7 subunit are under way to begin to understand the possible mechanisms.