RNA-mediated displacement of an inhibitory snRNP complex activates transcription elongation - PubMed (original) (raw)

RNA-mediated displacement of an inhibitory snRNP complex activates transcription elongation

Iván D'Orso et al. Nat Struct Mol Biol. 2010 Jul.

Abstract

The transition from transcription initiation to elongation at the HIV-1 promoter is controlled by Tat, which recruits P-TEFb to TAR RNA to phosphorylate RNA polymerase II. It has long been unclear why the HIV-1 promoter is incompetent for elongation. We report that P-TEFb is recruited to the promoter in a catalytically inactive state bound to the inhibitory 7SK small nuclear ribonucleoprotein (snRNP), thereby preventing elongation. It also has long been believed that TAR functions to recruit Tat to the promoter, but we find that Tat is recruited to the DNA template before TAR is synthesized. We propose that TAR binds Tat and P-TEFb as it emerges on the nascent transcript, competitively displacing the inhibitory 7SK snRNP and activating the P-TEFb kinase. Recruitment of an inhibitory snRNP complex at an early stage in the transcription cycle provides a new paradigm for controlling gene expression with a noncoding RNA.

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Figures

Figure 1

Figure 1

Inactive P-TEFb assembles into Pol II complexes and Tat activates its catalytic activity. (a) Partial composition of Pol II complexes purified on a TFIIS affinity resin was assessed by comparing Western blots of proteins and levels of 7SK snRNA eluted from a GST-TFIIS column to those from HeLa whole cell extracts (WCE) and a GST control column. The TFIIS used was an N-terminal fragment that possesses the same activity as full-length TFIIS. The purified coomassie-stained recombinant GST and GST-TFIIS proteins used for the columns are shown on the left. The arrow indicates the position of full-length CycT1. (b) GST-TFIIS eluted complexes were loaded onto a Sepharose CL-2B gel filtration column and the single eluted peak was immunoprecipitated (IP) by either anti-Cdk9 or mock (normal rabbit IgG) antibodies. Both precipitates, either incubated or not with purified GST-Tat, were used in kinase assays using a GST-CTD substrate and analyzed by Western blot with the H5 antibody, which recognizes Pol IIo. (c) Pol II complexes were purified on a TFIIS affinity resin, incubated with GST or GST-Tat in vitro, immunoprecipitated with anti-Cdk9 antibody, and the presence of P-TEFb (CycT1 and Cdk9) and 7SK snRNP (Hexim1 and Larp7) was monitored by Western blot.

Figure 2

Figure 2

Distribution of Tat and cofactors at the HIV-1 promoter and dependence on TAR. (a) Schematic of the HIV-1 LTR promoter-luciferase (FFL) reporter integrated into a HeLa cell line, showing the locations of the upstream region (−845), enhancer elements (−352), core promoter containing Sp1 and TATA-boxes (−75), transcription start site (TSS; +1), TAR element, FFL coding region, stop codon, and polyadenylation signal (p(A)). The locations of eight amplicons used in PCR quantification of ChIP-enriched DNA are shown. Numbers indicate the positions of the central base pair of each amplicon relative to the TSS. (b) ChIP assays were performed with protein extracts from the reporter cell line 48 hr after a mock (grey bars) or Flag-tagged Tat transfection (black bars) using the antibodies indicated. Values represent the percentage of input DNA immunoprecipitated and are the average of four independent PCRs from two separate immunoprecipitations from two independent cell cultures. All standard deviations are <15%. (c) ChIP assays were performed as in panel b but using extracts pre-treated with RNase A. (d) ChIP assays were performed with protein extracts obtained from a HeLa HIV-1 LTRΔTAR-FFL cell line 48 hr after a mock (grey bars) or Flag-tagged Tat transfection (black bars) using the antibodies indicated.

Figure 3

Figure 3

Tat assembles with the 7SK snRNP in vivo. RNA immunoprecipitation (RIP) was performed using antibodies to Flag-tagged Tat. Western blots (upper panels) indicate that components of P-TEFb (CycT1 and Cdk9) and 7SK snRNP (Larp7 and Hexim1) form a complex with Tat. The two lower panels utilized RT-PCR to detect 7SK and U6 snRNAs.

Figure 4

Figure 4

7SK snRNP and Tat recruitment to HIV-1 PICs are Sp1-dependent. (a) Promoterless and full-length LTR templates were immobilized to streptavidin-coated magnetic beads through a biotin moiety at the 5’ end, and a NotI site was used to cleave the promoter and elute proteins from the beads, which were then analyzed by Western blot. The activity of the purified HIV-1 PICs was measured by in vitro transcription and primer extension and yielded the expected transcription product (Tp), and was inhibited by α-amanitin treatment. (b) The immobilized full-length HIV-1 LTR, core, and mutant templates shown were incubated with HeLa nuclear extracts and washed, and proteins were eluted with NotI and identified by Western blot. In vitro transcription of the PICs produced the correct Tp. (c) HeLa HIV-1 LTR reporter cells were transfected with a control scrambled siRNA or a siRNA against Sp1, resulting in >80% depletion of Sp1 and decrease in reporter activity (Supplementary Fig. 5), and ChIP assays were performed using the antibodies indicated. Values represent the percentage of input DNA immunoprecipitated and are the average of four independent PCRs from two separate immunoprecipitations from two independent cell cultures. All standard deviations are <15%

Figure 5

Figure 5

Proposed model of HIV-1 transcription activation by Tat. (a) Tat assembles into complexes with P-TEFb (CycT1 and Cdk9) and the 7SK snRNP (Hexim1, Larp7, and 7SK snRNA). This Tat-7SK snRNP complex is recruited to HIV-1 PICs containing the Pol IIa form and the basal transcription machinery (e.g. Sp1, TBP), among other possible promoter-specific factors, and remains bound in the paused state (Paused complex). As transcription proceeds and TAR is synthesized, Tat facilitates the transfer of P-TEFb to the nascent RNA site. We propose that this Tat-TAR binding event competitively displaces 7SK snRNP and activates Cdk9 to phosphorylate Ser2 residues in the CTD (P) and assemble competent transcription elongation complexes containing a Pol IIo form. Hexim1 may dissociate from Larp7/7SK snRNA complexes, as it is not stably bound, and may be replaced by hnRNP proteins in a transcription-dependent manner,. (b) In the absence of TAR, Tat and P-TEFb do not transfer to the nascent RNA and evict the 7SK snRNP, preventing Ser2-CTD phosphorylation (P) and formation of elongation complexes.

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