P-TEFb regulation of transcription termination factor Xrn2 revealed by a chemical genetic screen for Cdk9 substrates.
P-TEFb regulation of transcription termination factor Xrn2 revealed by a chemical genetic screen for Cdk9 substrates.
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CDK9底物的化学遗传筛选显示了转录终止因子XRN2的P-TEFB调节。
DOI：
10.1101/gad.269589.115
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发表时间：
2016-01-01
影响因子：
10.5
通讯作者：
Fisher RP
中科院分区：
文献类型：
--
作者：
Sansó M;Levin RS;Lipp JJ;Wang VY;Greifenberg AK;Quezada EM;Ali A;Ghosh A;Larochelle S;Rana TM;Geyer M;Tong L;Shokat KM;Fisher RP
Sansóet al.鉴定了人正转录延伸因子b(P-TEFb)的RNA100假定底物，它富含与转录和∼分解代谢有关的蛋白质。在被CDK9磷酸化的RNA加工因子中，5‘-to-3’“鱼雷”外切核酸酶Xrn2是Pol II终止转录所必需的。RNA聚合酶II(Pol II)的转录周期在离散的转换点上由细胞周期蛋白依赖性蛋白激酶(CDKs)调节。正转录延伸因子b(P-TEFb)是一种CDK9和细胞周期蛋白T1的复合体，可促进暂停的Pol II的释放进入延长，但CDK9的确切作用机制和靶点仍不清楚。在这里，通过化学遗传策略，我们确定了人P-TEFb100的假定底物，这些底物富含与转录和∼分解代谢有关的蛋白质。在被CDK9磷酸化的RNA加工因子中，5‘到3’“鱼雷”外切核酸酶Xrn2是Pol II终止转录所必需的，我们在体内和体外验证了它是真正的P-TEFb底物。CDK9的磷酸化或其目标残基Thr439的仿磷取代在体外增强了Xrn2在合成底物上的酶活性。相反，抑制或耗尽CDK9或将Xrn2-Thr439突变为非磷酸化的Ala残基会导致与人类细胞低效终止一致的表型：Xrn2染色质定位受损，内源基因的直读转录增加。因此，除了在延伸中的作用外，P-TEFb还通过促进染色质招募和共转录RNA处理酶Xrn2的激活来调节终止。
Sansó et al. identified ∼100 putative substrates of human positive transcription elongation factor b (P-TEFb), which were enriched for proteins implicated in transcription and RNA catabolism. Among the RNA processing factors phosphorylated by Cdk9 was the 5′-to-3′ “torpedo” exoribonuclease Xrn2, required in transcription termination by Pol II. The transcription cycle of RNA polymerase II (Pol II) is regulated at discrete transition points by cyclin-dependent kinases (CDKs). Positive transcription elongation factor b (P-TEFb), a complex of Cdk9 and cyclin T1, promotes release of paused Pol II into elongation, but the precise mechanisms and targets of Cdk9 action remain largely unknown. Here, by a chemical genetic strategy, we identified ∼100 putative substrates of human P-TEFb, which were enriched for proteins implicated in transcription and RNA catabolism. Among the RNA processing factors phosphorylated by Cdk9 was the 5′-to-3′ “torpedo” exoribonuclease Xrn2, required in transcription termination by Pol II, which we validated as a bona fide P-TEFb substrate in vivo and in vitro. Phosphorylation by Cdk9 or phosphomimetic substitution of its target residue, Thr439, enhanced enzymatic activity of Xrn2 on synthetic substrates in vitro. Conversely, inhibition or depletion of Cdk9 or mutation of Xrn2-Thr439 to a nonphosphorylatable Ala residue caused phenotypes consistent with inefficient termination in human cells: impaired Xrn2 chromatin localization and increased readthrough transcription of endogenous genes. Therefore, in addition to its role in elongation, P-TEFb regulates termination by promoting chromatin recruitment and activation of a cotranscriptional RNA processing enzyme, Xrn2.