Garavís Miguel, González-Polo Noelia, Allepuz-Fuster Paula, Louro Jaime Alegrio, Fernández-Tornero Carlos, Calvo Olga
Instituto de Biología Funcional y Genómica. CSIC/Universidad de Salamanca, C/ Zacarías González 2, Salamanca 37007, Spain.
Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain.
Nucleic Acids Res. 2017 Mar 17;45(5):2458-2471. doi: 10.1093/nar/gkw1206.
Biogenesis of messenger RNA is critically influenced by the phosphorylation state of the carboxy-terminal domain (CTD) in the largest RNA polymerase II (RNAPII) subunit. Several kinases and phosphatases are required to maintain proper CTD phosphorylation levels and, additionally, several other proteins modulate them, including Rpb4/7 and Sub1. The Rpb4/7 heterodimer, constituting the RNAPII stalk, promote phosphatase functions and Sub1 globally influences CTD phosphorylation, though its mechanism remains mostly unknown. Here, we show that Sub1 physically interacts with the RNAPII stalk domain, Rpb4/7, likely through its C-terminal region, and associates with Fcp1. While Rpb4 is not required for Sub1 interaction with RNAPII complex, a fully functional heterodimer is required for Sub1 association to promoters. We also demonstrate that a complete CTD is necessary for proper association of Sub1 to chromatin and to the RNAPII. Finally, genetic data show a functional relationship between Sub1 and the RNAPII clamp domain. Altogether, our results indicate that Sub1, Rpb4/7 and Fcp1 interaction modulates CTD phosphorylation. In addition, Sub1 interaction with Rpb4/7 can also modulate transcription start site selection and transcription elongation rate likely by influencing the clamp function.
信使核糖核酸（mRNA）的生物合成受到最大的RNA聚合酶II（RNAPII）亚基中羧基末端结构域（CTD）磷酸化状态的关键影响。需要多种激酶和磷酸酶来维持适当的CTD磷酸化水平，此外，还有其他几种蛋白质对其进行调节，包括Rpb4/7和Sub1。构成RNAPII柄的Rpb4/7异二聚体促进磷酸酶功能，而Sub1对CTD磷酸化具有全局性影响，但其机制大多仍不为人知。在这里，我们表明Sub1可能通过其C末端区域与RNAPII柄结构域Rpb4/7发生物理相互作用，并与Fcp1相关联。虽然Rpb4不是Sub1与RNAPII复合物相互作用所必需的，但Sub1与启动子的关联需要一个功能完整的异二聚体。我们还证明，完整的CTD对于Sub1与染色质和RNAPII的正确关联是必需的。最后，遗传学数据显示Sub1与RNAPII钳结构域之间存在功能关系。总之，我们的结果表明Sub1、Rpb4/7和Fcp1的相互作用调节CTD磷酸化。此外，Sub1与Rpb4/7的相互作用还可能通过影响钳功能来调节转录起始位点的选择和转录延伸率。
