Brogie John E, Price David H
Biochemistry Department, University of Iowa, Iowa City, IA 52242, USA.
Nucleic Acids Res. 2017 Jun 20;45(11):6864-6880. doi: 10.1093/nar/gkx262.
The 7SK small nuclear ribonucleoprotein (snRNP) plays a central role in RNA polymerase II elongation control by regulating the availability of active P-TEFb. We optimized conditions for analyzing 7SK RNA by SHAPE and demonstrated a hysteretic effect of magnesium on 7SK folding dynamics including a 7SK GAUC motif switch. We also found evidence that the 5΄ end pairs alternatively with two different regions of 7SK giving rise to open and closed forms that dictate the state of the 7SK motif. We then used recombinant P-TEFb, HEXIM1, LARP7 and MEPCE to reconstruct a functional 7SK snRNP in vitro. Stably associated P-TEFb was highly inhibited, but could still be released and activated by HIV-1 Tat. Notably, P-TEFb association with both in vitro-reconstituted and cellular snRNPs led to similar changes in SHAPE reactivities, confirming that 7SK undergoes a P-TEFb-dependent structural change. We determined that the xRRM of LARP7 binds to the 3΄ stem loop of 7SK and inhibits the methyltransferase activity of MEPCE through a C-terminal MEPCE interaction domain (MID). Inhibition of MEPCE is dependent on the structure of the 3΄ stem loop and the closed form of 7SK RNA. This study provides important insights into intramolecular interactions within the 7SK snRNP.
7SK小核核糖核蛋白（snRNP）通过调节活性P-TEFb的可用性，在RNA聚合酶II延伸控制中发挥核心作用。我们优化了通过SHAPE分析7SK RNA的条件，并证明了镁对7SK折叠动力学的滞后效应，包括7SK GAUC基序开关。我们还发现证据表明，7SK的5΄端与7SK的两个不同区域交替配对，产生开放和封闭形式，决定了7SK基序的状态。然后，我们使用重组P-TEFb、HEXIM1、LARP7和MEPCE在体外重建了功能性7SK snRNP。稳定结合的P-TEFb受到高度抑制，但仍可被HIV-1 Tat释放和激活。值得注意的是，P-TEFb与体外重组和细胞snRNP的结合导致SHAPE反应性发生类似变化，证实7SK经历了依赖于P-TEFb的结构变化。我们确定LARP7的xRRM与7SK的3΄茎环结合，并通过C端MEPCE相互作用结构域（MID）抑制MEPCE的甲基转移酶活性。对MEPCE的抑制取决于3΄茎环的结构和7SK RNA的封闭形式。这项研究为7SK snRNP内的分子内相互作用提供了重要见解。
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