Raf1 Is a DCAF for the Rik1 DDB1-like protein and has separable roles in siRNA generation and chromatin modification.
Raf1 Is a DCAF for the Rik1 DDB1-like protein and has separable roles in siRNA generation and chromatin modification.
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DOI：
10.1371/journal.pgen.1002499
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发表时间：
2012-02
期刊：
影响因子：
4.5
通讯作者：
Allshire RC
中科院分区：
文献类型：
--
作者：
Buscaino A;White SA;Houston DR;Lejeune E;Simmer F;de Lima Alves F;Diyora PT;Urano T;Bayne EH;Rappsilber J;Allshire RC
非编码转录可以触发组蛋白翻译后修饰，形成特殊的染色质。在裂殖酵母中，异染色质的形成需要 RNAi 和组蛋白 H3K9 甲基转移酶复合物 CLRC（由 Clr4、Raf1、Raf2、Cul4 和 Rik1 组成）。 CLRC介导H3K9甲基化和siRNA产生；它还在体外显示 E3-泛素连接酶活性。 DCAF 作为 E3 连接酶的底物受体，可能将泛素化与组蛋白甲基化偶联。在这里，Raf1 中特征 WDxR 基序的结构比对和突变表明它是 CLRC 的 DCAF。我们证明 Raf1 通过两个不同的、可分离的功能促进 H3K9 甲基化和 siRNA 扩增。 DCAF Raf1 与 Cul4-Rik1 的关联对于 H3K9 甲基化至关重要，但对于将着丝粒转录物加工成 siRNA 来说却是可有可无的。因此，组蛋白甲基化和允许 RNAi 向染色质发出信号需要 DCAF、Raf1 与其接头 Rik1 的结合。异染色质是染色质的一种特殊形式，通常组装在几乎没有或没有编码潜力的 DNA 序列上。异染色质的形成涉及组蛋白尾部的特定翻译后修饰（例如组蛋白 H3 在赖氨酸 9 上的甲基化）。在裂殖酵母（裂殖酵母）中，异染色质存在于着丝粒、端粒和交配型基因座处。着丝粒的异染色质完整性对于正常的染色体分离很重要。已知裂殖酵母着丝粒处的异染色质相关重复序列会被转录，并且这些非编码转录物被加工成 siRNA。 H3K9 甲基化的建立和维持需要 siRNA 的产生。但 H3K9 甲基化本身是 siRNA 生产所必需的。目前尚不清楚这两个过程是如何耦合的。在本研究中，我们使用结构建模和遗传分析来证明异染色质成分 Raf1 在耦合 H3K9 甲基化和 siRNA 产生中发挥重要作用。我们的分析表明，异染色质因子 Rik1 和 Raf1 可以分别与 Cul4-E3 泛素连接酶组分 DDB1 和 DDB2 进行结构比对。我们发现特定的突变会损害 Raf1 与 Rik1 的关联并阻止 H3K9 甲基化，但不会阻止 siRNA 的产生。这些功能研究为 siRNA 生产和染色质修饰如何整合提供了机制见解。
Non-coding transcription can trigger histone post-translational modifications forming specialized chromatin. In fission yeast, heterochromatin formation requires RNAi and the histone H3K9 methyltransferase complex CLRC, composed of Clr4, Raf1, Raf2, Cul4, and Rik1. CLRC mediates H3K9 methylation and siRNA production; it also displays E3-ubiquitin ligase activity in vitro. DCAFs act as substrate receptors for E3 ligases and may couple ubiquitination with histone methylation. Here, structural alignment and mutation of signature WDxR motifs in Raf1 indicate that it is a DCAF for CLRC. We demonstrate that Raf1 promotes H3K9 methylation and siRNA amplification via two distinct, separable functions. The association of the DCAF Raf1 with Cul4-Rik1 is critical for H3K9 methylation, but dispensable for processing of centromeric transcripts into siRNAs. Thus the association of a DCAF, Raf1, with its adaptor, Rik1, is required for histone methylation and to allow RNAi to signal to chromatin. Heterochromatin is a specialized form of chromatin which is frequently assembled on DNA sequences with little or no coding potential. Heterochromatin formation involves specific post-translational modifications of histone tails (e.g. methylation of histone H3 on lysine 9). In fission yeast, Schizosaccharomyces pombe, heterochromatin is found at centromeres, telomeres, and the mating type locus. Heterochromatin integrity at centromeres is important for normal chromosome segregation. The heterochromatin associated repeats at fission yeast centromeres are known to be transcribed, and these non-coding transcripts are processed into siRNAs. siRNA production is required for establishment and maintenance of H3K9 methylation. But H3K9 methylation itself is required for siRNA production. It is not known how these two processes are coupled. In this study we use structural modelling and genetic analyses to demonstrate that the heterochromatin component Raf1 plays an essential role in coupling H3K9 methylation and siRNA production. Our analyses show that the heterochromatin factors Rik1 and Raf1 can be structurally aligned with Cul4-E3 ubiquitin ligase components DDB1 and DDB2, respectively. We show that specific mutations impair the association of Raf1 with Rik1 and prevent H3K9 methylation but not siRNA production. These functional studies provide mechanistic insights into how siRNA production and chromatin modification are integrated.
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影响因子：
11.4
作者：
Djupedal, Ingela;Kos-Braun, Isabelle C.;Ekwall, Karl
通讯作者：
Ekwall, Karl
影响因子：
4
作者：
Grewal, Shiv I. S.
通讯作者：
Grewal, Shiv I. S.
影响因子：
64.5
作者：
Braun S;Garcia JF;Rowley M;Rougemaille M;Shankar S;Madhani HD
通讯作者：
Madhani HD
影响因子：
56.9
作者：
Kagansky, Alexander;Folco, Hernan Diego;Allshire, Robin C.
通讯作者：
Allshire, Robin C.
DOI：
10.1073/pnas.0511160103
发表时间：
2006-02-21
影响因子：
11.1
作者：
Kapetanaki, MG;Guerrero-Santoro, J;Levine, AS
通讯作者：
Levine, AS