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 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 α-amanitin-resistant mutants of Pol II (CTD-52 and CTD-5; 10 μg of each). α-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 α-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 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 [γ-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 (α-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).

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 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 α-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).

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 [α-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 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 (α-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 [γ³²P]ATP.

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).