Subunits of the Drosophila CCR4-NOT complex and their roles in mRNA deadenylation
Subunits of the Drosophila CCR4-NOT complex and their roles in mRNA deadenylation
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DOI：
10.1261/rna.2145110
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发表时间：
2010-07-01
期刊：
影响因子：
4.5
通讯作者：
Wahle, Elmar
中科院分区：
文献类型：
--
作者：
Temme, Claudia;Zhang, Lianbing;Wahle, Elmar
CCR 4-NOT复合物是催化mRNA去腺苷化的主要酶。我们已经调查了这个复杂的果蝇的组成，通过免疫沉淀与针对NOT 1的单克隆抗体。CCR 4、CAF 1（= POP 2）、NOT 1、NOT 2、NOT 3和CAF 40亚基在稳定的复合物中缔合，但NOT 4不是。已知参与mRNA调控的因子在与CCR 4-NOT复合物共沉淀的其他蛋白质中是突出的，如通过质谱分析的。该复合物主要位于细胞质中，但似乎不是P体的主要成分。在已知的CCR 4旁系同源物中，发现Nocturnin与CCR 4-NOT复合物的亚基相关，而Angel和3635则不相关。在Schneider细胞中的RNAi实验表明，CAF 1、NOT 1、NOT 2和NOT 3是大量poly（A）缩短和hsp 70 mRNA去腺苷化所必需的，但CCR 4、CAF 40和NOT 4的敲低并不影响这些过程。过表达催化死亡的CAF 1对mRNA衰变有显性负效应。相反，过表达失活的CCR 4没有影响。我们得出结论，CAF 1是果蝇Schneider细胞中CCR 4-NOT复合物的主要催化重要亚基。Nocturnin也可能参与mRNA去腺苷化，而没有证据表明Angel和3635具有类似的作用。
The CCR4-NOT complex is the main enzyme catalyzing the deadenylation of mRNA. We have investigated the composition of this complex in Drosophila melanogaster by immunoprecipitation with a monoclonal antibody directed against NOT1. The CCR4, CAF1 (=POP2), NOT1, NOT2, NOT3, and CAF40 subunits were associated in a stable complex, but NOT4 was not. Factors known to be involved in mRNA regulation were prominent among the other proteins coprecipitated with the CCR4-NOT complex, as analyzed by mass spectrometry. The complex was localized mostly in the cytoplasm but did not appear to be a major component of P bodies. Of the known CCR4 paralogs, Nocturnin was found associated with the subunits of the CCR4-NOT complex, whereas Angel and 3635 were not. RNAi experiments in Schneider cells showed that CAF1, NOT1, NOT2, and NOT3 are required for bulk poly(A) shortening and hsp70 mRNA deadenylation, but knock-down of CCR4, CAF40, and NOT4 did not affect these processes. Overexpression of catalytically dead CAF1 had a dominant-negative effect on mRNA decay. In contrast, overexpression of inactive CCR4 had no effect. We conclude that CAF1 is the major catalytically important subunit of the CCR4-NOT complex in Drosophila Schneider cells. Nocturnin may also be involved in mRNA deadenylation, whereas there is no evidence for a similar role of Angel and 3635.