The mechanism of release of P-TEFb and HEXIM1 from the 7SK snRNP by viral and cellular activators includes a conformational change in 7SK.
The mechanism of release of P-TEFb and HEXIM1 from the 7SK snRNP by viral and cellular activators includes a conformational change in 7SK.
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DOI：
10.1371/journal.pone.0012335
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发表时间：
2010-08-23
期刊：
影响因子：
3.7
通讯作者：
Price DH
中科院分区：
文献类型：
--
作者：
Krueger BJ;Varzavand K;Cooper JJ;Price DH
正转录延伸因子P-TEFb是产生mRNAs所必需的，但大部分因子存在于7SK SnRNP中，在那里它被HEXIM1灭活。在体内，HIV-1Tat的表达导致P-TEFb和HEXIM1从7SK SnRNP释放，但释放机制尚不清楚。我们建立了一种体外P-TEFb释放实验，用LARP7抗体从HeLa细胞裂解物中免疫沉淀7SK SnRNP，并将其与潜在的释放因子孵育。我们发现，P-TEFb可通过HIV-1 TAT或细胞激活剂Brd4的P-TEFb结合区直接从7SK SnRNP释放。甘油梯度沉淀法分析表明，相同的Brd4蛋白导入HeLa细胞后，7SK SnRNP体内可释放P-TEFb和HEXIM1。虽然HEXIM1在体外与7SK RNA紧密结合，但从7SK SnRNP释放P-TEFb伴随着HEXIM1的丢失。利用化学修饰的方法，我们确定伴随着HEXIM1的释放，7SK经历了主要的构象变化，阻止了HEXIM1的重新结合。鉴于启动子近端暂停的聚合酶存在于大多数人类基因上，了解激活剂如何将P-TEFb招募到这些基因是至关重要的。我们的结果表明，两种被测试的激活剂都可以从7SK SnRNP中提取P-TEFb。重要的是，我们发现在提取P-TEFb后，伴随着HEXIM1的排出，7SK发生了显著的构象变化。根据我们的发现，我们假设HEXIM1重新整合到7SK SnRNP中可能是含有P-TEFb的7SK SnRNP的重组调控步骤。
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. 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. 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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