Mechanisms of Transcription Laboratory, Clare Hall Laboratories, Cancer Research UK London Research Institute, Blanche Lane, South Mimms, Hertfordshire EN6 3LD, United Kingdom.
Proc Natl Acad Sci U S A. 2009 Dec 8;106(49):20705-10. doi: 10.1073/pnas.0907052106. Epub 2009 Nov 17.
The proteasome degrades proteins modified by polyubiquitylation, so correctly controlled ubiquitylation is crucial to avoid unscheduled proteolysis of essential proteins. The mechanism regulating proteolysis of RNAPII has been controversial since two distinct ubiquitin ligases (E3s), Rsp5 (and its human homologue NEDD4) and Elongin-Cullin complex, have both been shown to be required for its DNA-damage-induced polyubiquitylation. Here we show that these E3s work sequentially in a two-step mechanism. First, Rsp5 adds mono-ubiquitin, or sometimes a ubiquitin chain linked via ubiquitin lysine 63 that does not trigger proteolysis. When produced, the K63 chain can be trimmed to mono-ubiquitylation by an Rsp5-associated ubiquitin protease, Ubp2. Based on this mono-ubiquitin moiety on RNAPII, an Elc1/Cul3 complex then produces a ubiquitin chain linked via lysine 48, which can trigger proteolysis. Likewise, for correct polyubiquitylation of human RNAPII, NEDD4 cooperates with the ElonginA/B/C-Cullin 5 complex. These data indicate that RNAPII polyubiquitylation requires cooperation between distinct, sequentially acting ubiquitin ligases, and raise the intriguing possibility that other members of the large and functionally diverse family of NEDD4-like ubiquitin ligases also require the assistance of a second E3 when targeting proteins for degradation.
蛋白酶体降解多泛素化修饰的蛋白质，因此正确控制泛素化对于避免必需蛋白质的非计划性降解至关重要。自从两种不同的泛素连接酶（E3）——Rsp5（及其人类同源物 NEDD4）和 Elongin-Cullin 复合物——都被证明需要参与 DNA 损伤诱导的 RNAPII 多泛素化以来，调节 RNAPII 蛋白水解的机制一直存在争议。在这里，我们展示了这些 E3 在两步机制中依次发挥作用。首先，Rsp5 添加单泛素，或者有时添加通过泛素赖氨酸 63 连接的泛素链，而这种连接不会引发蛋白水解。当产生时，K63 链可以被 Rsp5 相关的泛素蛋白酶 Ubp2 修剪为单泛素化。基于 RNAPII 上的这种单泛素部分，Elc1/Cul3 复合物随后产生通过赖氨酸 48 连接的泛素链，这可以触发蛋白水解。同样，对于人 RNAPII 的正确多泛素化，NEDD4 与 ElonginA/B/C-Cullin 5 复合物合作。这些数据表明，RNAPII 的多泛素化需要不同的、依次作用的泛素连接酶之间的合作，并提出了一个有趣的可能性，即其他具有大而多样化功能的 NEDD4 样泛素连接酶家族成员在将蛋白质靶向降解时也需要第二种 E3 的协助。
