The Ccr4-Not complex regulates essentially every aspect of gene expression, from mRNA synthesis to protein destruction. The Not4 subunit of the complex contains an E3 RING domain and targets proteins for ubiquitin-dependent proteolysis. Ccr4-Not associates with elongating RNA polymerase II (RNAPII), which raises the possibility that it controls the degradation of elongation complex components. Here, we demonstrate that Ccr4-Not controls the ubiquitylation and turnover of Rpb1, the largest subunit of RNAPII, during transcription arrest. Deleting NOT4 or mutating its RING domain strongly reduced the DNA damage-dependent ubiquitylation and destruction of Rpb1. Surprisingly, in vitro ubiquitylation assays indicate that Ccr4-Not does not directly ubiquitylate Rpb1 but instead promotes Rpb1 ubiquitylation by the HECT domain-containing ligase Rsp5. Genetic analyses suggest that Ccr4-Not acts upstream of RSP5, where it acts to initiate the destruction process. Ccr4-Not binds Rsp5 and forms a ternary complex with it and the RNAPII elongation complex. Analysis of mutant Ccr4-Not lacking the RING domain of Not4 suggests that it both recruits Rsp5 and delivers the E2 Ubc4/5 to RNAPII. Our work reveals a previously unknown function of Ccr4-Not and identifies an essential new regulator of RNAPII turnover during genotoxic stress.

中文翻译：

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Ccr4-Not通过控制被捕的RNAPII的泛素化和降解来维持基因组完整性。

从mRNA合成到蛋白质破坏，Ccr4-Not复合物基本上调节基因表达的各个方面。该复合物的Not4亚基包含一个E3 RING结构域，并针对泛素依赖性蛋白水解作用靶向蛋白质。Ccr4-Not与延伸RNA聚合酶II（RNAPII）相关，这增加了它控制延伸复合物组分降解的可能性。在这里，我们证明了Ccr4-Not在转录逮捕期间控制着Rpb1（RNAPII的最大亚基）的泛素化和转换。删除NOT4或使其RING结构域突变会大大减少依赖于DNA损伤的Rpb1泛素化和破坏。出人意料的是，体外泛素化测定表明，Ccr4-Not不会直接泛化Rpb1，而是通过含HECT结构域的连接酶Rsp5促进Rpb1泛素化。遗传分析表明，Ccr4-Not在RSP5的上游起作用，在该处引发破坏过程。Ccr4-Not与Rsp5结合并与Rsp5和RNAPII延伸复合物形成三元复合物。不缺少Not4 RING结构域的突变体Ccr4-的分析表明，它既募集Rsp5，又将E2 Ubc4 / 5递送至RNAPII。我们的工作揭示了Ccr4-Not以前未知的功能，并确定了在遗传毒性胁迫下RNAPII转换的必要新调节剂。