Temme Claudia, Zaessinger Sophie, Meyer Sylke, Simonelig Martine, Wahle Elmar
Institut für Biochemie, Martin-Luther-Universität Halle-Wittenberg, Halle, Germany.
EMBO J. 2004 Jul 21;23(14):2862-71. doi: 10.1038/sj.emboj.7600273. Epub 2004 Jun 24.
The CCR4-NOT complex is the major enzyme catalyzing mRNA deadenylation in Saccharomyces cerevisiae. We have identified homologs for almost all subunits of this complex in the Drosophila genome. Biochemical fractionation showed that the two likely catalytic subunits, CCR4 and CAF1, were associated with each other and with a poly(A)-specific 3' exonuclease activity. In Drosophila, the CCR4 and CAF1 proteins were ubiquitously expressed and present in cytoplasmic foci. Individual knock-down of several potential subunits of the Drosophila CCR4-NOT complex by RNAi in tissue culture cells led to a lengthening of bulk mRNA poly(A) tails. Knock-down of two individual subunits also interfered with the rapid deadenylation of Hsp70 mRNA during recovery from heat shock. Similarly, ccr4 mutant flies had elongated bulk poly(A) and a defect in Hsp70 mRNA deadenylation. A minor increase in bulk poly(A) tail length was also observed in Rga mutant flies, which are affected in the NOT2 subunit. The data show that the CCR4-NOT complex is conserved in Drosophila melanogaster and plays a role in general and regulated mRNA deadenylation.
CCR4-NOT复合物是酿酒酵母中催化mRNA去腺苷酸化的主要酶。我们已在果蝇基因组中鉴定出该复合物几乎所有亚基的同源物。生化分级分离表明，两个可能的催化亚基CCR4和CAF1彼此相关，并具有聚腺苷酸特异性3'外切核酸酶活性。在果蝇中，CCR4和CAF1蛋白广泛表达并存在于细胞质聚集体中。在组织培养细胞中通过RNAi分别敲低果蝇CCR4-NOT复合物的几个潜在亚基，导致大量mRNA聚腺苷酸尾巴延长。敲低两个亚基也会干扰热休克恢复过程中Hsp70 mRNA的快速去腺苷酸化。同样，ccr4突变果蝇的大量聚腺苷酸延长，并且在Hsp70 mRNA去腺苷酸化方面存在缺陷。在受NOT2亚基影响的Rga突变果蝇中也观察到大量聚腺苷酸尾巴长度略有增加。数据表明，CCR4-NOT复合物在黑腹果蝇中保守，并在一般和受调控的mRNA去腺苷酸化中起作用。
