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. 1999 Sep;19(9):5960-8.
doi: 10.1128/MCB.19.9.5960.

Tat-SF1 protein associates with RAP30 and human SPT5 proteins

J B Kim  1 , Y Yamaguchi, T Wada, H Handa, P A Sharp
Affiliations

Tat-SF1 protein associates with RAP30 and human SPT5 proteins

J B Kim et al. Mol Cell Biol. 1999 Sep.

Abstract

The potent transactivator Tat recognizes the transactivation response RNA element (TAR) of human immunodeficiency virus type 1 and stimulates the processivity of elongation of RNA polymerase (Pol) II complexes. The cellular proteins Tat-SF1 and human SPT5 (hSPT5) are required for Tat activation as shown by immunodepletion with specific sera and complementation with recombinant proteins. In nuclear extracts, small fractions of both hSPT5 and Pol II are associated with Tat-SF1 protein. Surprisingly, the RAP30 protein of the heterodimeric transcription TFIIF factor is associated with Tat-SF1, while the RAP74 subunit of TFIIF is not coimmunoprecipitated with Tat-SF1. Overexpression of Tat-SF1 and hSPT5 specifically stimulates the transcriptional activity of Tat in vivo. These results suggest that Tat-SF1 and hSPT5 are indispensable cellular factors supporting Tat-specific transcription activation and that they may interact with RAP30 in controlling elongation.

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Figures

FIG. 1
FIG. 1
Tat-SF1 associates with RAP30 of TFIIF. (A) Tat-SF1 coimmunoprecipitated with RAP30. HeLa total cell extracts (250 μg) were used for immunoprecipitation with antibodies against preimmune (PI, lane 2), Tat-SF1 (lane 3) (44), and RAP30 of TFIIF (lane 4) (Santa Cruz Biotechnology). The input lane contained 20% of the extract used for the immunoprecipitation (lane 1). Western blots of the immunoprecipitates were probed with anti-Tat-SF1 (top) and anti-RAP30 (bottom) antibodies. (B) Tat-SF1-depleted HeLa nuclear extracts contained decreased levels of RAP30. HeLa nuclear extracts were immunodepleted with anti-Tat-SF1 antibody. After each depletion step, aliquots were taken and Western blotting was performed with antibodies against Tat-SF1 and RAP30. Anti-Cdk9 antibody and anti-RAP74 antibodies (Santa Cruz Biotechnology) were used for the control.
FIG. 2
FIG. 2
Tat-SF1 interacts with hSPT5 and Pol II. (A) Tat-SF1 coimmunoprecipitated with hSPT5 and Pol II. HeLa total cell extracts were immunoprecipitated with antibodies against preimmune (lane 1), Tat-SF1 (lane 2), and hSPT5 (lane 3). Western blots of immunoprecipitates were probed with anti-Pol II CTD (top) (38), anti-hSPT5 (middle) (31), and anti-Tat-SF1 (bottom) antibodies. (B) Expression of hSPT5 in vivo increases the level of association with Tat-SF1. HeLa cells were transfected with vector control (mock) or hSPT5 expression vectors. Aliquots of total cell extracts were immunoprecipitated with anti-Tat-SF1 antibody, and immunoprecipitates were probed with antibodies against Pol II CTD (top), hSPT5 (middle), and Tat-SF1 (bottom) for Western blotting. (C) Isolation of f-Tat-SF1 and its associated protein complexes. An extract of HeLa cells transfected with f-Tat-SF1 was incubated with anti-Flag beads and washed as described in Materials and Methods. As a control, mock-transfected extract was prepared (lanes 1 and 2). To elute bound complex, Flag peptides (∼3.75 μg) were incubated with the beads. After each round of elution, aliquots of eluted fractions were separated by SDS-PAGE, and the Western blots were developed with antibodies against Pol II CTD, hSPT5, Flag (Sigma), and RAP30 of TFIIF.
FIG. 3
FIG. 3
Tat-SF1 and hSPT5 are required for Tat-dependent activation. (A) Analysis of Tat-SF1- or hSPT5-depleted nuclear extracts with Western blotting. HeLa nuclear extracts were immunodepleted four times with preimmune (PI) (lane 2), anti-hSPT5 (lane 3), and anti-Tat-SF1 (lane 4) antibodies. Aliquots of each depleted extract were probed with antibodies against Pol II CTD, hSPT5, Tat-SF1, RAP30, and TBP. An equal volume (2 μl) of input nuclear extract was used for the control (lane 1). (B) Scheme of the Tat-dependent TAR-specific transcription reaction. HeLa nuclear extracts were preincubated with template DNAs including pHIV-LTR+TAR and pHIV-LTRΔTAR at 30°C for 30 min. Radiolabelled CTP and 200 μM of cold nucleotide mixtures (ATP, GTP, CTP, and UTP) were added into the reactions for transcription elongation. RNase T1 digestion was carried out to distinguish the Tat-dependent TAR-specific transcripts (+TAR G400) versus the TAR-independent transcripts (ΔTAR G100). (C) Tat-SF1 depleted- and hSPT5 depleted-nuclear extracts are defective for Tat-dependent activation. Immunodepleted nuclear extracts (ΔTat-SF1, lanes 3 and 4; ΔhSPT5, lanes 5 and 6), as well as control nuclear extracts (lanes 1 and 2), were used for transcription in vitro. Purified Tat protein (∼25 ng) was added during the preincubation step as indicated (lanes 2, 4, and 6). TAR-specific transcription activities were calculated by quantitation of the synthesized transcripts from +TAR/ΔTAR. Fold levels of Tat-specific transcription activities were obtained by normalization of TAR-specific transcription activities in the presence or absence of Tat protein as described previously (20). (D) Depletion of Tat-SF1 and hSPT5 did not change the transcription activities from the adenovirus E4 promoter and the MLP. In vitro transcription activities from each of the depleted nuclear extracts, as well as control nuclear extracts, were determined for E4 and MLP (see Materials and Methods).
FIG. 4
FIG. 4
Addition of recombinant Tat-SF1 and DSIF proteins rescues Tat-dependent transcription activation. (A) SDS-PAGE analysis of recombinant Tat-SF1 and DSIF proteins. Samples (∼250 ng) of purified recombinant Tat-SF1 (lane 1, open arrowhead) and DSIF, a complex of hSPT4 and hSPT5 (lane 2, closed arrowhead), were resolved by SDS-PAGE on a 4 to 20% gel, and the proteins were visualized by staining gels with Coomassie brilliant blue. (B) Normalization of baculovirus-expressed Tat-SF1. Full-length rTat-SF1 protein was purified from recombinant baculovirus-infected Hi5 cells. Then, 4 μg of HeLa nuclear extracts (lanes 1 and 2) and different amounts of baculovirus-expressed rTat-SF1 proteins (lanes 3 to 5) were analyzed by Western blotting. Approximately 50 ng of purified recombinant Tat-SF1 protein (lane 3) showed the same level of Tat-SF1 as 30 μg of HeLa nuclear extracts (4 μl). (C) Nuclear extracts depleted with Tat-SF1 and hSPT5 can be complemented by recombinant Tat-SF1 and DSIF proteins for Tat-dependent activation. Recombinant Tat-SF1 (lanes 3 and 4) or DSIF, a complex of hSPT4 and hSPT5 (lanes 7 and 8), proteins were added to reactions at the preincubation step. The fold activation by Tat was measured as described previously (20).
FIG. 5
FIG. 5
Ectopic expression of Tat-SF1 and hSPT5 specifically enhances Tat-dependent transcription activity in vivo. (A) Expression of Tat-SF1 and hSPT5 increased Tat-dependent transcription activity. HeLa cells were cotransfected with HIV-LTR CAT reporter DNA (1 μg), 1 μg of Tat-SF1 expression vector (see Materials and Methods), and/or 1 μg of hSPT5 expression vector (38) in the absence or presence of the Tat expression vector (50 ng) as indicated. (B) Expression of Tat-SF1 and hSPT5 did not increase the transcription activities by Gal4-VP16 in vivo. HeLa cells were cotransfected with UAS-CAT reporter (1 μg), 1 μg of Tat-SF1 expression vector, and/or 1 μg of hSPT5 expression vector in the absence or presence of Gal4-VP16 expression vector (200 ng). In both panels A and B, the relative CAT activities were obtained by normalization with β-Gal activities. pCMV–β-Gal expression vector (0.5 μg) was cotransfected in each transfection for the normalization of CAT assay. All transfection experiments were performed in duplicate and independently repeated five times. The results are representative of three separate experiments.
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