MAQ1 and 7SK RNA interact with CDK9/cyclin T complexes in a transcription-dependent manner

Abstract

Positive transcription elongation factor b (P-TEFb) comprises a cyclin (T1 or T2) and a kinase, cyclin-dependent kinase 9 (CDK9), which phosphorylates the carboxyl-terminal domain of RNA polymerase II. P-TEFb is essential for transcriptional elongation in human cells. A highly specific interaction among cyclin T1, the viral protein Tat, and the transactivation response (TAR) element RNA determines the productive transcription of the human immunodeficiency virus genome. In growing HeLa cells, half of P-TEFb is kinase inactive and binds to the 7SK small nuclear RNA. We now report on a novel protein termed MAQ1 (for ménage à quatre) that is also present in this complex. Since 7SK RNA is required for MAQ1 to associate with P-TEFb, a structural role for 7SK RNA is proposed. Inhibition of transcription results in the release of both MAQ1 and 7SK RNA from P-TEFb. Thus, MAQ1 cooperates with 7SK RNA to form a novel type of CDK inhibitor. According to yeast two-hybrid analysis and immunoprecipitations from extracts of transfected cells, MAQ1 binds directly to the N-terminal cyclin homology region of cyclins T1 and T2. Since Tat also binds to this cyclin T1 N-terminal domain and since the association between 7SK RNA/MAQ1 and P-TEFb competes with the binding of Tat to cyclin T1, we speculate that the TAR RNA/Tat lentivirus system has evolved to subvert the cellular 7SK RNA/MAQ1 system.

FIG. 1.

Selective coimmunoprecipitation of a 65-kDa protein with CDK9 and cyclin T1. (A) Lysates from ³⁵S-labeled HeLa cells were fractionated by ultracentrifugation. The large P-TEFb/7SK complex was found in gradient fraction 7 (−) unless transcription had been inhibited with actinomycin D (ActD) prior to lysis (+) (41). Gradient fraction 7 was immunoprecipitated with anti-cyclin T1 (lanes 1 and 2) or anti-CDK9 (lanes 3 and 4) antibodies or a control mock serum (lanes 5 and 6). (B) Lysates from G3H cells were fractionated by ultracentrifugation. P-TEFb was immunoprecipitated with protein A beads cross-linked to 12CA5 (anti-HA) antibodies (lanes 1 and 3) or control mock serum (lane 2) from gradient fraction 7. Lanes 1 and 2 correspond to an autoradiogram of the ³⁵S-labeled immunoprecipitates. Lane 3 is Coomassie blue staining of a preparative 12CA5 immunoprecipitate. The positions of cyclin T1, CDK9, and a coimmunoprecipitating 65-kDa protein (p65) are indicated.

FIG. 2.

Identification of MAQ1, a protein coded by the HEXIM1 RNA. (A) Amino acid sequence predicted from the HEXIM1 RNA sequence (32). Tryptic peptides identified in the MALDI/TOF spectrum are underlined. A putative nuclear localization sequence (bold) was predicted by the PredictNLS server (http://cubic.bioc.columbia.edu/predictNLS/). (B) HeLa cells transiently transfected (FlagMAQ1, lanes 2 and 4) or not transfected (control, lanes 1 and 3) with pAdRSV-FlagMAQ1 were lysed and analyzed by Western blotting for endogenous MAQ1 and Flag-MAQ1 by anti-MAQ1 (lanes 1 and 2) or anti-Flag antibodies (lanes 3 and 4). (C) Alignment of human, mouse, chicken, zebra fish, and drosophila MAQ protein sequences deduced from cDNAs in the GenBank database. Accession numbers are given in parentheses. The Homo sapiens MAQ1 (Hs1; AB021179), H. sapiens MAQ1 paralogue (Hs2; AK056946), Mus musculus MAQ1 (Mm1; AY090614), M. musculus MAQ1 orthologue to Hs2 (Mm2; BC026458), G. gallus MAQ1 orthologue (Gg; BU488401 and BU374772), D. renio MAQ1 orthologue (Dr; BG307670), and Drosophila melanogaster MAQ1 orthologue (Dm; AY051786) sequences are shown. The human MAQ1 amino acid numbering is indicated above the sequences. The dark boxes enclose residues that are conserved in other species.

FIG. 3.

Transcription-dependent association of MAQ1 with P-TEFb/7SK RNA complexes. (A) Lysates, from cells treated (lane 2) or not treated (lane 1) with 1 μg of actinomycin D ml⁻¹ for 1 h were immunoprecipitated with anti-cyclin T1 (lanes 5 and 6), anti-MAQ1 (lanes 7 and 8), or preimmune (mock) antiserum (lanes 3 and 4). Cyclin T1, MAQ1, and CDK9 in inputs and immunoprecipitates (beads) were analyzed by Western blotting. (B) Lysates from cells treated (Act D) or not treated (control) with actinomycin D were immunoprecipitated with anti-MAQ1 and analyzed for 7SK and U4 RNA by Northern blotting. I, inputs; S, supernatants; B, beads. Preimmune serum was used as a negative control (mock). (C) Cells were treated or not treated (control) with 10 μM DRB and lysed after 1 h (+), or DRB was washed out and the cells were allowed to recover for another hour in fresh medium before lysis (+/−). Alternatively, cells were irradiated at 254 nm (UV) and lysed immediately (lane 0) or allowed to recover for 1 h at 37°C (lane 1). Cyclin T1, MAQ1, and CDK9 retained on beads by anti-MAQ1 were detected by Western blotting.

FIG. 4.

MAQ1 is a subunit of P-TEFb/7SK RNA complexes. (A) Fractions from glycerol gradients. Gradients were loaded with a lysate from HeLa cells treated (ActD) or not treated (untreated) with 1 μg of actinomycin D ml⁻¹. Alternatively, RNase A was added to the lysate from untreated cells (RNase). (B) Supernatants (S) or protein A beads (B) after immunoprecipitation (IP) with either preimmune (mock) or MAQ1 antiserum from gradient fractions containing either core P-TEFb (fraction 3) or P-TEFb/7SK complexes (fraction 7). (C) Cell lysates treated (+) or not treated (−) with 500 mM NaCl or RNase A were immunoprecipitated with preimmune serum (mock), MAQ1 antiserum (MAQ1), or anti-cyclin T1 (T1). Samples were analyzed for cyclin T1 and MAQ1 by Western blotting.

FIG. 5.

Requirements for MAQ1 association with P-TEFb. (A) HeLa cells were transfected with carrier plasmid (control) or expression vectors for HA-tagged full-length cyclin T1, cyclin T2b, cyclin T2a, CDK9, or CDK9_D167N (CDK9mut). Cells were treated (ActD) or not treated (untreated) with actinomycin D. Lysates were immunoprecipitated with protein A beads and anti-HA antibodies. Notably, the cyclin T2b cDNA generated both cyclin T2a and T2b proteins because of alternative splicing (44). (B) HeLa cells were transfected with the carrier plasmid (control) or expression vectors for HA-tagged full-length cyclin T1 (wt T1) and C-terminal deletion mutants CycT1(1-333) and CycT1(1-254). Cells were treated (+) or not treated (−) with actinomycin D. Lysates were immunoprecipitated with protein A beads and anti-HA antibodies. (C) HeLa cells were transfected with the carrier plasmid (control) or expression vectors for Flag-tagged full-length MAQ1 (wt MAQ1), C-terminal deletion mutants MAQ1(1-180) and MAQ1(1-240), or N-terminal deletion mutants MAQ1(120-359) and MAQ1(181-359). Transfected cells were treated (+) or not treated (−) with actinomycin D and lysed. Lysates were immunoprecipitated with protein A beads and anti-Flag antibodies. Transfected epitope-tagged proteins (input), endogenous MAQ1 protein, and endogenous cyclin T1 were detected by Western blotting with appropriate antibodies. 7SK RNA was detected by Northern blotting. Positions of molecular size markers are indicated.

FIG. 6.

MAQ1 is a nuclear protein. (A) Immunofluorescence of HeLa cells stained with anti-MAQ1 antibodies (left) or 4′,6′-diamidino-2-phenylindole (DAPI; right). (B) HeLa cells transiently transfected with full-length Flag-MAQ1 (wt) or deletion mutants Flag-MAQ1(1-180), Flag-MAQ1(1-240) Flag-MAQ1(120-359), and Flag-MAQ1(181-359). Immunofluorescence assay with anti-Flag antibodies is shown on the left. Chromatin staining with DAPI is shown on the right.

FIG. 7.

The MAQ1 C-terminal domain directly interacts with the N-terminal domain of cyclins T1 and T2. The interactions of MAQ1 with CDK9 and cyclin T were analyzed with a yeast two-hybrid system coexpressing LexA DNA-binding domain fusion proteins with GAL4 activation domain fusion proteins. β-Galactosidase activities (in arbitrary units [a.u.]) were quantified in pools of more than 10 yeast double transformants. Fusion protein combinations are described on the left. A, full-length proteins; B, cyclin T deletion mutants; C, MAQ1 deletion mutants.

FIG. 8.

P-TEFb/7SK RNA/MAQ1 complexes are impaired for Tat binding. (A) The small core active P-TEFb complex (glycerol gradient fraction 3) and the large inactive P-TEFb complex (glycerol gradient fraction 7) were tested for the ability to associate with GST-Tat72, GST-Tat72_K41, and GST-Tat48. Cyclin T1 and MAQ1 were probed by Western blotting, and 7SK and U4 RNAs were probed by Northern blotting. (B) GST-Tat72 was tested for the ability to bind cyclin T1 from gradient fraction 3 or 7 after addition (+) or no addition (−) of NaCl up to 500 mM. (C) GST-Tat48 was tested for the ability to bind cyclin T1 from gradient fraction 3 or 7 after addition (+) or no addition (−) of RNase A. Inputs corresponded to 25% of the fractions incubated with GST-Tat.