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. 2007 Aug 8;26(15):3570-80.
doi: 10.1038/sj.emboj.7601783. Epub 2007 Jul 5.

Dynamic remodelling of human 7SK snRNP controls the nuclear level of active P-TEFb

Elodie Van Herreweghe  1 , Sylvain Egloff, Isabelle Goiffon, Beáta E Jády, Carine Froment, Bernard Monsarrat, Tamás Kiss
Affiliations

Dynamic remodelling of human 7SK snRNP controls the nuclear level of active P-TEFb

Elodie Van Herreweghe et al. EMBO J. .

Abstract

The 7SK small nuclear RNA (snRNA) regulates RNA polymerase II transcription elongation by controlling the protein kinase activity of the positive transcription elongation factor b (P-TEFb). In cooperation with HEXIM1, the 7SK snRNA sequesters P-TEFb into the kinase-inactive 7SK/HEXIM1/P-TEFb small nuclear ribonucleoprotein (snRNP), and thereby, controls the nuclear level of active P-TEFb. Here, we report that a fraction of HeLa 7SK snRNA that is not involved in 7SK/HEXIM1/P-TEFb formation, specifically interacts with RNA helicase A (RHA), heterogeneous nuclear ribonucleoprotein A1 (hnRNP), A2/B1, R and Q proteins. Inhibition of cellular transcription induces disassembly of 7SK/HEXIM1/P-TEFb and at the same time, increases the level of 7SK snRNPs containing RHA, hnRNP A1, A2/B1, R and Q. Removal of transcription inhibitors restores the original levels of the 7SK/HEXIM1/P-TEFb and '7SK/hnRNP' complexes. 7SK/HEXIM1/P-TEFb snRNPs containing mutant 7SK RNAs lacking the capacity for binding hnRNP A1, A2, R and Q are resistant to stress-induced disassembly, indicating that recruitment of the novel 7SK snRNP proteins is essential for disruption of 7SK/HEXIM1/P-TEFb. Thus, we propose that the nuclear level of active P-TEFb is controlled by dynamic and reversible remodelling of 7SK snRNP.

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Figures

Figure 1
Figure 1
Detection of HeLa proteins interacting with 7SK snRNA in vitro. (A) Structures of 7SK test RNAs. The secondary structure of human 7SK snRNA has been adopted (Wassarman and Steitz, 1991). The boundaries of internal deletions d1–d7, introduced into the 7SKd1–7SKd7 RNAs, are indicted by arrows. The F1, F2, F3 and F4 fragments of 7SK snRNA are boxed. (B) In vitro RNA–protein crosslinking. In vitro synthesized internally labelled 7SK RNA was incubated with a HeLa nuclear extract. Formation of covalent RNA–protein interactions was induced by irradiation with UV light. After RNase treatment, proteins marked by label transfer were separated on a 12% SDS–polyacrylamide gel and detected by autoradiography. E. coli tRNA (lanes 2–4) or cold 7SK snRNA (lanes 6–8) were added in 10- (lanes 2 and 6), 100- (lanes 3 and 7) and 1000-fold (lanes 4 and 8) excess. The stability of 7SK probe RNAs was verified (lower panels). Protein and DNA size markers are indicated. (C) In vitro crosslinking of internally truncated 7SK RNAs. (D) In vitro crosslinking of 7SK snRNA fragments. (E) In vitro crosslinking of 7SK snRNA in the presence of 10- (lanes 2, 6, 10 and 14) 100- (lanes 3, 7, 11 and 15) or 1000-fold (4, 8, 12 and 16) excess of cold F1, F2, F3 or F4 RNAs.
Figure 2
Figure 2
Identification of HeLa proteins associated with 7SK RNA. In vitro assembled 7SK RNP particles were affinity-selected with a biotinylated antisense oligoribonucleotide immobilized on streptavidin agarose beads. Proteins associated with beads in the absence (mock) or presence of 7SK RNA were analysed by SDS–PAGE. Proteins specific for 7SK-containing beads were isolated and subjected to mass spectrometry. Minor proteins bands indicated by asterisks correspond to *hnRNP U, **DRBP76 and ***hnRNP C1. IP of HeLa hnRNP C1 or transiently expressed HA-tagged DRBP76 and hnRNP U proteins failed to recover significant amounts of 7SK snRNA (data not shown).
Figure 3
Figure 3
In vivo association of 7SK snRNA with RHA and hnRNP A1, A2/B1, R and Q proteins. (A) Transient expression of MS2-7SK RNA. A schematic representation of MS2-7SK RNA with highlighted MS2-binding sequences is shown. From a sonic extract of HeLa cells expressing the MS2-7SK RNA and HA-tagged HEXIM1, snRNPs were immunoprecipitated with anti-CycT1 (α-CT1) and anti-HA (α-HA) antibodies. RNAs extracted from the pellets were fractionated on a 6% sequencing gel, electroblotted onto a nylon membrane and probed with a mixture of labelled oligonucleotides specific for the human 7SK, U2 and MRP snRNAs. Lane Ex, RNAs from HeLa cell extract; lane M, size markers. (B) Affinity selection of MS2-7SK RNA with a recombinant MS2-MBP fusion protein. RNAs affinity-selected from HeLa cell extracts not expressing (mock) or expressing the MS2-7SK RNA were terminally labelled with cytidine 3′,5′-bis(phosphate) (pCp) and T4 RNA ligase and analysed on a 6% sequencing gel. Lane Ex, RNAs from HeLa cell extract expressing MS2-7SK. (C) Proteins associated with transiently expressed MS2-7SK RNA. Proteins co-purified with MS2-7SK RNA or mock-purified from a cell extract not expressing MS2-7SK (mock) were immunoblotted with antibodies as indicated on the right. Lane Ex, cell extract. Protein size markers are indicated. (D) Transient expression of epitope-tagged hnRNP proteins. Proteins extracted from HeLa cells either transfected (T) or not transfected (N) with expression vectors for hnRNP A2-V5, B1-V5, HA-R HA-Q1 and HA-Q2 were analysed by Western blotting with specific antibodies. (E) Proteins interacting with HeLa 7SK snRNA. RNAs co-immunoprecipitated with HeLa hnRNP A1 or with transiently expressed hnRNP A2-V5 (A2-V5), hnRNP B1-V5 (B1-V5), HA-hnRNP R (HA-R), HA-hnRNP Q1 (HA-Q1), HA-hnRNP Q2 (HA-Q2) and FL-RHA proteins were tested by Northern blotting. RNAs recovered from cell extracts (Ex) or immunoprecipitated with non-specific IgG (Ct) were also assayed. IP of proteins was verified by Western blot analysis (lower panels). (F) In vivo RNP-IP. Transfected (A2-V5, B1-V5) or non-transfected (A1) HeLa cells were cross-linked with formaldehyde. After extract preparation, hnRNP A1, A2-V5 and B1-V5 were immunoprecipitated under stringent conditions. Co-precipitation of 7SK and U2 snRNA was tested by RNase A/T1 mapping. RNAs extracted from one fiftieth of the extracts (Ex) or mock-precipitated with nonspecific IgG (Ct) were also mapped. IP of proteins was verified by Western blotting (lower panels).
Figure 4
Figure 4
7SK elements directing in vivo binding of hnRNP A1, A2, R and Q1. (A) Analysis of the protein binding capacity of internally truncated 7SK RNAs. Mutant 7SK RNAs, indicated above the lanes, were transiently expressed in HeLa cells also expressing epitope-tagged hnRNP A2-V5, HA-hnRNP R or HA-hnRNP Q1 proteins. Cellular sonic extracts were prepared and 7SK snRNPs were immunoprecipitated with anti-hnRNP A1, anti-HA or anti-V5 antibodies. RNAs extracted from the pellets (P) and supernatants (S) were analysed by Northern blotting with 7SK-specific oligonucleotide probes. Endogenous 7SK snRNA is shown. (B) 7SK-3HP RNA corresponding to the G196-C277 region of human 7SK snRNA interacts with hnRNP A1 and A2. 7SK-3HP RNA and hnRNP A2-V5 protein were transiently coexpressed in HeLa cells. RNAs purified from cell extract (Ex) or co-immunoprecipitated with hnRNP A1 (α-A1) or A2-V5 (α-V5) were subjected to Northern blot analysis. Lanes Ct, control IP with nonspecific IgG.
Figure 5
Figure 5
HeLa 7SK snRNA exists in multiple snRNPs. (A) Proteins associated with 7SK/HEXIM/P-TEFb snRNP. Proteins immunoprecipitated from a HeLa cell extract with an anti-CycT1 antibody (α-CT1) or with nonspecific IgG (Ct) were separated on a 12% SDS-polyacrylamide gel, electroblotted onto a nitrocellulose membrane and stained with antibodies indicated on the right. Lane Ex, cell extract. (B) HeLa hnRNP A1, hnRNP R and RHA do not associate with P-TEFb. Proteins co-immunoprecipitated with HeLa hnRNP A1 (α-A1) and transiently expressed HA-R (α-HA) and FL-RHA (α-FL) were characterized by Western blotting with antibodies indicated on the right.
Figure 6
Figure 6
Dynamic remodelling of HeLa 7SK snRNP. (A) ActD treatment of HeLa cells increases the nuclear levels of 7SK snRNPs containing RHA, hnRNP A1, A2, R and Q. Epitope-tagged hnRNP A2-V5, HA-hnRNP R, HA-hnRNP Q1 and FL-RHA proteins were transiently expressed in HeLa cells. RNAs phenol-extracted (Ext) or immunoprecipitated (antibodies are indicated above the lanes) from extracts prepared from ActD-treated (+) or non-treated (−) cells were analysed by Northern blotting (upper panels). The efficacy of IPs was monitored by Western blot analysis (lower panels). Lanes Ct, control IPs with nonspecific IgG; lanes M, size markers. (B) Relative association of HeLa 7SK snRNA with 7SK snRNP proteins in ActD-treated HeLa cells. The intensities of 7SK snRNAs detected by Northern hybridization were quantified by phosphorimager. The relative levels of 7SK were normalized to the snRNP proteins recovered by IP. The levels of 7SK RNAs immunoprecipitated from non-treated control cells were set as 100%. Standard deviations are indicated. (C) Dynamics of 7SK snRNP in DRB-treated HeLa cells. HeLa hnRNP A1 and transiently expressed hnRNP A2-V5, HA-hnRNP R, HA-hnRNP Q1 and FL-RHA were immunoprecipitated, and association of 7SK snRNA was monitored by Northern blot analysis. Extracts for IP were prepared immediately after administration (0 h), 1 h after administration (1 h) and 1 h after removal of DRB (2 h). The efficiency of IP was confirmed by Western blot analysis (lower panels).
Figure 7
Figure 7
The third hairpin of 7SK is required for ActD-induced disassembly of 7SK/HEXIM/P-TEFb. (A) 7SKd5 and 7SKd6 RNAs stably associate with P-TEFb. HeLa cells expressing 7SKd4, 7SKd5 or 7SKd6 RNAs were treated (+) or non-treated (−) with ActD for 1 h before extract preparation. RNAs phenol-extracted (Ext) or immunoprecipitated with an anti-CycT1 (α-CT1) antibody were analysed by Northern blotting. IP of CycT1 was verified by Western blot analysis (lower panel). (B) Relative association of 7SK, 7SKd4, 7SKd5 and 7SKd6 RNAs with CycT1 in ActD-treated and control cells. The relative levels of 7SK, 7SKd4, 7SKd5 and 7SKd6 RNAs immunoprecipitated from control cells were set as 100%. The expression constructs (p7SKd4, p7SKd5 and p7SKd6) used for transfection of HeLa cells are indicated.
Figure 8
Figure 8
A model for regulation of the nuclear level of active P-TEFb by dynamic and reversible remodelling of 7SK snRNPs. In exponentially growing HeLa cells, about 50% of P-TEFb is sequestered into the 7SK/HEXIM1/P-TEFb snRNP, while the other half associates with the bromodomain protein 4 (Brd4) that likely recruits active P-TEFb to chromatin templates. Transcription inhibition by ActD or DRB treatment induces dissociation of P-TEFb and HEXIM1 from the 7SK snRNA and at the same time, facilitates binding of RHA, hnRNP A1, A2/B1, R and Q proteins. Please note that two copies of HEXIM1 and P-TEFb interact with one 7SK molecule in the 7SK/HEXIM1/P-TEFb snRNP, and that at least two different 7SK/hnRNP particles with not yet fully clarified protein composition are formed upon transcription inhibition.
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