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The mechanism of release of P-TEFb and HEXIM1 from the 7SK snRNP by viral and cellular activators includes a conformational change in 7SK.

Krueger BJ, Varzavand K, Cooper JJ, Price DH - PLoS ONE (2010)

Bottom Line: We found that P-TEFb was directly released from the 7SK snRNP by HIV-1 Tat or the P-TEFb binding region of the cellular activator Brd4.Importantly, we found that after P-TEFb is extracted a dramatic conformational change occurred in 7SK concomitant with the ejection of HEXIM1.Based on our findings, we hypothesize that reincorporation of HEXIM1 into the 7SK snRNP is likely the regulated step of reassembly of the 7SK snRNP containing P-TEFb.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Cellular Biology Program, University of Iowa, Iowa City, Iowa, United States of America.

ABSTRACT

Background: The positive transcription elongation factor, P-TEFb, is required for the production of mRNAs, however the majority of the factor is present in the 7SK snRNP where it is inactivated by HEXIM1. Expression of HIV-1 Tat leads to release of P-TEFb and HEXIM1 from the 7SK snRNP in vivo, but the release mechanisms are unclear.

Methodology/principal findings: We developed an in vitro P-TEFb release assay in which the 7SK snRNP immunoprecipitated from HeLa cell lysates using antibodies to LARP7 was incubated with potential release factors. We found that P-TEFb was directly released from the 7SK snRNP by HIV-1 Tat or the P-TEFb binding region of the cellular activator Brd4. Glycerol gradient sedimentation analysis was used to demonstrate that the same Brd4 protein transfected into HeLa cells caused the release of P-TEFb and HEXIM1 from the 7SK snRNP in vivo. Although HEXIM1 binds tightly to 7SK RNA in vitro, release of P-TEFb from the 7SK snRNP is accompanied by the loss of HEXIM1. Using a chemical modification method, we determined that concomitant with the release of HEXIM1, 7SK underwent a major conformational change that blocks re-association of HEXIM1.

Conclusions/significance: Given that promoter proximally paused polymerases are present on most human genes, understanding how activators recruit P-TEFb to those genes is critical. Our findings reveal that the two tested activators can extract P-TEFb from the 7SK snRNP. Importantly, we found that after P-TEFb is extracted a dramatic conformational change occurred in 7SK concomitant with the ejection of HEXIM1. Based on our findings, we hypothesize that reincorporation of HEXIM1 into the 7SK snRNP is likely the regulated step of reassembly of the 7SK snRNP containing P-TEFb.

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The P-TEFb binding region of Brd4 causes a release of P-TEFb from the 7SK snRNP in vivo.HeLa cells were transfected with plasmids expressing Brd4 1209–1362 (Brd4) or mutant Brd4 1209–1362 Δ1329–1345 (Brd4Δ). A) Immunofluorescence microscopy. 48 hours after transfection cells were fixed and stained for DNA (DAPI) or the FLAG tagged Brd4 constructs (FLAG). B) Glycerol gradient sedimentation analysis. Cell lysates were prepared 48 hr after transfection and sedimented on 5–45% glycerol gradients as described in Materials and Methods. Western blots of fractions were probed with antibodies to LARP7 or Cdk9 as indicated.
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pone-0012335-g003: The P-TEFb binding region of Brd4 causes a release of P-TEFb from the 7SK snRNP in vivo.HeLa cells were transfected with plasmids expressing Brd4 1209–1362 (Brd4) or mutant Brd4 1209–1362 Δ1329–1345 (Brd4Δ). A) Immunofluorescence microscopy. 48 hours after transfection cells were fixed and stained for DNA (DAPI) or the FLAG tagged Brd4 constructs (FLAG). B) Glycerol gradient sedimentation analysis. Cell lysates were prepared 48 hr after transfection and sedimented on 5–45% glycerol gradients as described in Materials and Methods. Western blots of fractions were probed with antibodies to LARP7 or Cdk9 as indicated.

Mentions: To determine if the P-TEFb binding domain of Brd4 would cause the release of P-TEFb from the 7SK snRNP in vivo, HeLa cells were transfected with plasmids that led to the expression FLAG tagged versions of either the wildtype P-TEFb binding domain of Brd4 (1209–1362) or the mutant domain (1209–1362 Δ1329–1345). Transfection efficiencies were similar for the two proteins and greater than 50% as evidenced by immunofluorescence microscopy using antibodies against the FLAG epitope (Figure 3A). High levels of both proteins were found in the nucleus and cytoplasm (Figure 3A). Whole cell lysates were generated from mock transfected cells as well as the two Brd4 transfected cells and analyzed by glycerol gradient sedimentation. As expected, Western blot analysis of LARP7 and Cdk9 from the control cell lysate indicated that most of the P-TEFb was in the more rapidly sedimenting 7SK snRNP (Figure 3B, frax 10–12). An identical pattern was obtained from the Brd4Δ expressing cells (Figure 3B). However, expression of Brd4 (1209–1362) had a dramatic effect in that most of the P-TEFb was now found in slower sedimenting fractions (Figure 3B frax 4–6). As has been found before [51], LARP7 sedimentation was unaffected by release of P-TEFb. These in vivo findings support the in vitro results and strongly implicate Brd4 in the release of P-TEFb from the 7SK snRNP.


The mechanism of release of P-TEFb and HEXIM1 from the 7SK snRNP by viral and cellular activators includes a conformational change in 7SK.

Krueger BJ, Varzavand K, Cooper JJ, Price DH - PLoS ONE (2010)

The P-TEFb binding region of Brd4 causes a release of P-TEFb from the 7SK snRNP in vivo.HeLa cells were transfected with plasmids expressing Brd4 1209–1362 (Brd4) or mutant Brd4 1209–1362 Δ1329–1345 (Brd4Δ). A) Immunofluorescence microscopy. 48 hours after transfection cells were fixed and stained for DNA (DAPI) or the FLAG tagged Brd4 constructs (FLAG). B) Glycerol gradient sedimentation analysis. Cell lysates were prepared 48 hr after transfection and sedimented on 5–45% glycerol gradients as described in Materials and Methods. Western blots of fractions were probed with antibodies to LARP7 or Cdk9 as indicated.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2925947&req=5

pone-0012335-g003: The P-TEFb binding region of Brd4 causes a release of P-TEFb from the 7SK snRNP in vivo.HeLa cells were transfected with plasmids expressing Brd4 1209–1362 (Brd4) or mutant Brd4 1209–1362 Δ1329–1345 (Brd4Δ). A) Immunofluorescence microscopy. 48 hours after transfection cells were fixed and stained for DNA (DAPI) or the FLAG tagged Brd4 constructs (FLAG). B) Glycerol gradient sedimentation analysis. Cell lysates were prepared 48 hr after transfection and sedimented on 5–45% glycerol gradients as described in Materials and Methods. Western blots of fractions were probed with antibodies to LARP7 or Cdk9 as indicated.
Mentions: To determine if the P-TEFb binding domain of Brd4 would cause the release of P-TEFb from the 7SK snRNP in vivo, HeLa cells were transfected with plasmids that led to the expression FLAG tagged versions of either the wildtype P-TEFb binding domain of Brd4 (1209–1362) or the mutant domain (1209–1362 Δ1329–1345). Transfection efficiencies were similar for the two proteins and greater than 50% as evidenced by immunofluorescence microscopy using antibodies against the FLAG epitope (Figure 3A). High levels of both proteins were found in the nucleus and cytoplasm (Figure 3A). Whole cell lysates were generated from mock transfected cells as well as the two Brd4 transfected cells and analyzed by glycerol gradient sedimentation. As expected, Western blot analysis of LARP7 and Cdk9 from the control cell lysate indicated that most of the P-TEFb was in the more rapidly sedimenting 7SK snRNP (Figure 3B, frax 10–12). An identical pattern was obtained from the Brd4Δ expressing cells (Figure 3B). However, expression of Brd4 (1209–1362) had a dramatic effect in that most of the P-TEFb was now found in slower sedimenting fractions (Figure 3B frax 4–6). As has been found before [51], LARP7 sedimentation was unaffected by release of P-TEFb. These in vivo findings support the in vitro results and strongly implicate Brd4 in the release of P-TEFb from the 7SK snRNP.

Bottom Line: We found that P-TEFb was directly released from the 7SK snRNP by HIV-1 Tat or the P-TEFb binding region of the cellular activator Brd4.Importantly, we found that after P-TEFb is extracted a dramatic conformational change occurred in 7SK concomitant with the ejection of HEXIM1.Based on our findings, we hypothesize that reincorporation of HEXIM1 into the 7SK snRNP is likely the regulated step of reassembly of the 7SK snRNP containing P-TEFb.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Cellular Biology Program, University of Iowa, Iowa City, Iowa, United States of America.

ABSTRACT

Background: The positive transcription elongation factor, P-TEFb, is required for the production of mRNAs, however the majority of the factor is present in the 7SK snRNP where it is inactivated by HEXIM1. Expression of HIV-1 Tat leads to release of P-TEFb and HEXIM1 from the 7SK snRNP in vivo, but the release mechanisms are unclear.

Methodology/principal findings: We developed an in vitro P-TEFb release assay in which the 7SK snRNP immunoprecipitated from HeLa cell lysates using antibodies to LARP7 was incubated with potential release factors. We found that P-TEFb was directly released from the 7SK snRNP by HIV-1 Tat or the P-TEFb binding region of the cellular activator Brd4. Glycerol gradient sedimentation analysis was used to demonstrate that the same Brd4 protein transfected into HeLa cells caused the release of P-TEFb and HEXIM1 from the 7SK snRNP in vivo. Although HEXIM1 binds tightly to 7SK RNA in vitro, release of P-TEFb from the 7SK snRNP is accompanied by the loss of HEXIM1. Using a chemical modification method, we determined that concomitant with the release of HEXIM1, 7SK underwent a major conformational change that blocks re-association of HEXIM1.

Conclusions/significance: Given that promoter proximally paused polymerases are present on most human genes, understanding how activators recruit P-TEFb to those genes is critical. Our findings reveal that the two tested activators can extract P-TEFb from the 7SK snRNP. Importantly, we found that after P-TEFb is extracted a dramatic conformational change occurred in 7SK concomitant with the ejection of HEXIM1. Based on our findings, we hypothesize that reincorporation of HEXIM1 into the 7SK snRNP is likely the regulated step of reassembly of the 7SK snRNP containing P-TEFb.

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