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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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Model of P-TEFb and HEXIM1 release from the 7SK snRNP.P-TEFb is directly extracted from the 7SK snRNP by Tat or Brd4. This leads to the loss of P-TEFb, a destabilization of the 7SK structure resulting in a conformational change in the RNA that causes HEXIM1 to be released from the 7SK snRNP. hnRNP proteins then bind to the region of RNA unmasked by the loss of HEXIM1.
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pone-0012335-g006: Model of P-TEFb and HEXIM1 release from the 7SK snRNP.P-TEFb is directly extracted from the 7SK snRNP by Tat or Brd4. This leads to the loss of P-TEFb, a destabilization of the 7SK structure resulting in a conformational change in the RNA that causes HEXIM1 to be released from the 7SK snRNP. hnRNP proteins then bind to the region of RNA unmasked by the loss of HEXIM1.

Mentions: Based on our results here and on published studies, we propose a more refined model for release of P-TEFb from the 7SK snRNP (Figure 6). Tat, Brd4 or another P-TEFb extractor binds directly to P-TEFb and removes it from the complex. This leaves HEXIM1 bound to the 7SK snRNP. In the second step, HEXIM1 is released from the RNA and 7SK rearranges into an alternative conformation. Loss of HEXIM1 could be caused by the rearrangement of 7SK or the rearrangement could occur after HEXIM1 dissociates. What is clear is that the equilibrium between free and RNA bound HEXIM1 lies strongly in favor of free HEXIM1 after P-TEFb is lost from the complex. P-TEFb has affinity for 7SK and HIV TAR RNA [37], [48] and it is possible that this helps stabilize the binding of HEXIM1 to 7SK in the presence of P-TEFb. After P-TEFb and HEXIM1 are released the 7SK snRNP associates with a number of hnRNP proteins [51], [52], [54], [60]. Presumably this further stabilizes the P-TEFb/HEXIM-free 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)

Model of P-TEFb and HEXIM1 release from the 7SK snRNP.P-TEFb is directly extracted from the 7SK snRNP by Tat or Brd4. This leads to the loss of P-TEFb, a destabilization of the 7SK structure resulting in a conformational change in the RNA that causes HEXIM1 to be released from the 7SK snRNP. hnRNP proteins then bind to the region of RNA unmasked by the loss of HEXIM1.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2925947&req=5

pone-0012335-g006: Model of P-TEFb and HEXIM1 release from the 7SK snRNP.P-TEFb is directly extracted from the 7SK snRNP by Tat or Brd4. This leads to the loss of P-TEFb, a destabilization of the 7SK structure resulting in a conformational change in the RNA that causes HEXIM1 to be released from the 7SK snRNP. hnRNP proteins then bind to the region of RNA unmasked by the loss of HEXIM1.
Mentions: Based on our results here and on published studies, we propose a more refined model for release of P-TEFb from the 7SK snRNP (Figure 6). Tat, Brd4 or another P-TEFb extractor binds directly to P-TEFb and removes it from the complex. This leaves HEXIM1 bound to the 7SK snRNP. In the second step, HEXIM1 is released from the RNA and 7SK rearranges into an alternative conformation. Loss of HEXIM1 could be caused by the rearrangement of 7SK or the rearrangement could occur after HEXIM1 dissociates. What is clear is that the equilibrium between free and RNA bound HEXIM1 lies strongly in favor of free HEXIM1 after P-TEFb is lost from the complex. P-TEFb has affinity for 7SK and HIV TAR RNA [37], [48] and it is possible that this helps stabilize the binding of HEXIM1 to 7SK in the presence of P-TEFb. After P-TEFb and HEXIM1 are released the 7SK snRNP associates with a number of hnRNP proteins [51], [52], [54], [60]. Presumably this further stabilizes the P-TEFb/HEXIM-free 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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