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. 2006 Jan;26(2):630-42.
doi: 10.1128/MCB.26.2.630-642.2006.

Regulation of polymerase II transcription by 7SK snRNA: two distinct RNA elements direct P-TEFb and HEXIM1 binding

Sylvain Egloff  1 , Elodie Van Herreweghe, Tamás Kiss
Affiliations

Regulation of polymerase II transcription by 7SK snRNA: two distinct RNA elements direct P-TEFb and HEXIM1 binding

Sylvain Egloff et al. Mol Cell Biol. 2006 Jan.

Abstract

The positive transcription elongation factor b (P-TEFb), a complex of Cdk9 and cyclin T1/T2, stimulates transcription by phosphorylating RNA polymerase II. The 7SK small nuclear RNA, in cooperation with HEXIM1 protein, functions as a general polymerase II transcription regulator by sequestering P-TEFb into a large kinase-inactive 7SK/HEXIM1/P-TEFb complex. Here, determination and characterization of the functionally essential elements of human 7SK snRNA directing HEXIM1 and P-TEFb binding led to a new model for the assembly of the 7SK/HEXIM1/P-TEFb regulatory complex. We demonstrate that two structurally and functionally distinct protein binding elements located in the 5'- and 3'-terminal hairpins of 7SK support the in vivo recruitment of HEXIM1 and P-TEFb. Consistently, a minimal regulatory RNA composed of the 5' and 3' hairpins of 7SK can modulate polymerase II transcription in HeLa cells. HEXIM1 binds independently and specifically to the G24-C48/G60-C87 distal segment of the 5' hairpin of 7SK. Binding of HEXIM1 is a prerequisite for association of P-TEFb with the G302-C324 apical region of the 3' hairpin of 7SK that is highly reminiscent of the human immunodeficiency virus transactivation-responsive RNA.

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Figures

FIG. 1.
FIG. 1.
Two distinct regions of 7SK snRNA are required for in vivo binding of P-TEFb. (A) Structures of transiently expressed internally truncated 7SK snRNAs. The schematic structure of the p7SK expression construct and the proposed secondary structure of human 7SK snRNA (31) are shown. The 7SK coding region (open arrow), the transcription initiation site (open arrowhead), and the relevant restriction sites (P, PstI; E, EcoRI) are indicated. Internal deletions are indicated by arrows (d1 to d11) or shaded boxes (d12 to d14). (B) Localization of P-TEFb binding elements in 7SK snRNA. Mutant 7SK snRNAs, indicated above the lanes, were transiently expressed in G3H cells expressing an HA-tagged version of CycT1 (21). Cellular extracts were incubated with protein A-agarose saturated with anti-HA antibody. After centrifugation, RNAs extracted from the agarose beads (P) and supernatants (S) were fractionated in 6% denaturing polyacrylamide gels and probed with terminally labeled oligonucleotides complementary to the human 7SK snRNA. The endogenous wild-type 7SK snRNA is shown (7SK). As controls, RNAs immunoselected from nontransfected G3H (G) and HeLa (N) cells were also probed. Lane C represents a control blot with E. coli tRNA that was used to facilitate RNA precipitation. Lane M, size markers in nucleotides (terminally labeled HaeIII- and TaqI-digested pBR322). (C) Identification of P-TEFb binding elements in the 5′-terminal hairpin of 7SK snRNA. For details, see the legend to panel B.
FIG. 2.
FIG. 2.
Elements directing HEXIM1 binding are confined to the distal portion of the 5′-terminal hairpin of 7SK. (A) Expression of HA-tagged HEXIM1. Accumulation of HA-HEXIM1 in HeLa cells either transfected (H) or untransfected (N) with the pHA-HEXIM1 expression construct was monitored by Western blotting with an anti-HA antibody. Molecular mass markers (kDa) are shown. (B) Determination of 7SK snRNA regions supporting in vivo binding of HEXIM1. (C) Mapping of HEXIM1 binding elements in the 5′ hairpin of 7SK. Details are given in the legend to Fig. 1.
FIG. 3.
FIG. 3.
Expression of 7SK5′+3′hp represses luciferase expression controlled by the SV40 promoter. (A) Expression of 7SK5′hp and 7SK5′+3′hp RNAs. The predicted schematic structures of 7SK5′hp and 7SK5′+3′hp RNAs coexpressed with HA-HEXIM1 in HeLa cells or with HA-P-TEFb in G3H cells are indicated. Interaction of 7SK5′hp and 7SK5′+3′hp RNAs with HA-HEXIM1 and HA-P-TEFb was tested by co-IP followed by Northern analysis. For other details, see the legend to Fig. 1. (B) In situ localization of 7SK5′+3′hp. HeLa cells transfected or untransfected (control) with p7SK5′+3′hp were hybridized with a fluorescent oligonucleotide probe complementary to the junction region of the 5′ and 3′ hairpins of 7SK5′+3′hp. Wild-type 7SK snRNA was localized in untransfected HeLa cells by using a sequence-specific fluorescent probe. Nuclei were stained with DAPI. The bars represent 10 μm. (C) Luciferase gene expression driven by the SV40 promoter. HeLa cells were cotransfected with the pSV4LUC luciferase reporter plasmid and the indicated expression and control plasmids. The cells were lysed in reporter lysis buffer, and the protein content and luciferase activity were determined. The activities of lysates obtained from cells transfected with pSV4LUC and pBST were set as 100%. The average values (with standard deviations) of six independent experiments, each performed in quintuplicate, are shown. (D) RNA analysis. RNAs were isolated from HeLa cells cotransfected with pSV4LUC and the indicated plasmids. Accumulation of luciferase mRNA (LUC), the RNA Pol III-transcribed U6-5.8 transfection control RNA, and the endogenous U6 snRNA was measured by RNase A/T1 mapping. Lane C represents control mapping with untransfected HeLa RNAs. Accumulation of 7SK, 7SKd12, and 7SK5′-3′hp was monitored by Northern blot analysis. (E) Relative accumulation of ectopically expressed 7SK, 7SKd12, and 7SK5′+3′hp. The intensities of 7SK RNAs were quantified by phosphorimager. Overaccumulation of the wild-type 7SK snRNA was determined by subtraction of the level of endogenous 7SK measured in control cells transfected with pBST. The relative levels of 7SK, 7SKd12, and 7SK5′+3′hp were normalized to the transiently expressed U6-5.8 RNA. The relative accumulation of ectopically expressed 7SK was considered 100%. Standard deviations are indicated.
FIG. 4.
FIG. 4.
Evolutionary conservation of 7SK elements required for HEXIM1 and P-TEFb binding. (A) Alignment of vertebrate 7SK snRNA sequences. The sequences of human (GenBank accession number NR_001445), rat (K02909), and lamprey (Lampetra fluviatilis) (11) 7SK snRNAs have been reported. The sequences of chicken (AJ890101), zebra fish (AJ890102), Tetraodon nigrovidis (AJ890103), and Fugu rubripes (AJ890104) 7SK RNAs were identified in our laboratory (unpublished results). Invariant nucleotides are indicated in red, while nucleotides conserved in all but one sequence are in blue. The 5′- and 3′-hairpin areas and regions essential for HEXIM1 and P-TEFb binding are indicated. (B) Proposed secondary structures of human 7SK elements directing HEXIM1 and P-TEFb binding. The proposed stem (H1, H2, H3, H4, Hd, and Hp) and internal-loop (IL) structures are indicated. The nucleotide changes in vertebrate 7SK RNAs are shown.
FIG. 5.
FIG. 5.
Structural requirements of HEXIM1 and P-TEFb binding to the 5′-terminal hairpin of human 7SK snRNA. (A) Sequence alterations introduced into the 5′-hairpin region of the p7SK expression construct. The proposed helices (H1 to H4) and the internal loop (IL) are indicated. (B) Interactions of transiently expressed mutant 7SK RNAs with HA-tagged HEXIM1 and P-TEFb. The transiently expressed and endogenous 7SK RNAs were detected by RNase A/T1 mapping with antisense RNA probes specific for the ectopically expressed mutant 7SK RNAs. For other details, see the legends to Fig. 1 and 2.
FIG. 6.
FIG. 6.
Elements directing P-TEFb binding to the 3′ hairpin of 7SK snRNA. (A) Structures of modified 7SK RNAs assayed for P-TEFb binding ability. Deleted (d17, d18, and d19) and altered sequences are shown. (B) Association of internally truncated 7SK RNAs with HA-P-TEFb. The endogenous (7SK) and transiently expressed 7SK RNAs were detected by Northern analysis. (C) Association of 7SK RNAs carrying altered 3′-hairpin sequences with HA-P-TEFb. 7SK RNAs were detected by RNase protection analysis performed with probes complementary to the mutant RNAs.
FIG. 7.
FIG. 7.
Elements directing in vivo binding of HEXIM1 and P-TEFb are crucial for in vitro assembly of 7SK/HEXIM1/P-TEFb and in vivo inhibition of Pol II transcription. (A) In vitro reconstitution of 7SK/HEXIM1/P-TEFb particle. From a G3H cell extract, P-TEFb and HA-P-TEFb were immobilized on protein A-agarose beads either coated (+) with an anti-CycT1 (a-CT1) antibody or uncoated (−). The beads were incubated with purified His-tagged HEXIM1 protein (His-H1) and in vitro-transcribed labeled 7SK RNAs as indicated. Retention of HA-CycT1 (HA-CT1), His-HEXIM1 (His-H1), and 7SK RNAs was tested by Western blot analysis and polyacrylamide gel electrophoresis, respectively. (D) In vivo inhibition of luciferase gene expression with coexpression of mutant 7SK RNAs. For details, see the legend to Fig. 3.
FIG. 8.
FIG. 8.
7SK-dependent regulation of the CTD kinase activity of P-TEFb. (A) Structural organization of the kinase-inactive 7SK/HEXIM1/P-TEFb complex. The schematic structure of human 7SK snRNA, together with associated proteins, is shown. Our experiments do not exclude the possibility that HEXIM1 binds to 7SK snRNA as a homodimer (39). (B) Structural comparison of the HIV TAR RNA and the P-TEFb binding motif of the 3′ hairpin of human 7SK snRNA. The conserved nucleotides are circled. Nucleotides essential for Tat binding are shaded (13). The “inverted Tat binding-like” motif of 7SK snRNA is boxed.
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