Reconstitution of Targeted Deadenylation by the Ccr4-Not Complex and the YTH Domain Protein Mmi1.
Reconstitution of Targeted Deadenylation by the Ccr4-Not Complex and the YTH Domain Protein Mmi1.
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DOI：
10.1016/j.celrep.2016.10.066
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发表时间：
2016-11-15
期刊：
影响因子：
8.8
通讯作者：
Passmore LA
中科院分区：
文献类型：
--
作者：
Stowell JAW;Webster MW;Kögel A;Wolf J;Shelley KL;Passmore LA
Ccr 4-Not是一种保守的蛋白复合物，它缩短真核生物mRNA的3′ poly（A）尾，调节转录物的稳定性和翻译成蛋白质。RNA结合蛋白被认为促进Ccr 4-Not向某些mRNA的募集，但缺乏体外重建系统减缓了理解这种特异性的机制细节的进展。在这里，我们产生了一个完全重组的Ccr 4-Not复合物，从RNA底物中去除poly（A）尾。完整的复合物比单独的核酸外切酶更有活性，并且对某些RNA具有内在的偏好。RNA结合蛋白Mmi 1在天然Ccr 4-Not的制备物中高度丰富。我们证明了重组Ccr 4-Not和Mmi 1之间的高亲和力相互作用。使用体外测定，我们表明，Mmi 1加速靶RNA的去腺苷化。总之，我们的研究结果支持了一个模型，即RNA结合蛋白和mRNA的序列背景影响去腺苷化速率来调节基因表达。我们纯化了一个完全重组的粟酒裂殖酵母Ccr 4-Not复合物。该复合物具有内在的底物偏好性，Ccr 4和Caf 1都是活性的Ccr 4-Not与YTH结构域RNA结合蛋白Mmi 1稳定地相互作用，Mmi 1以序列特异性方式加速Ccr 4-Not对RNA的去腺苷酸化。Poly（A）尾调节mRNA的稳定性和翻译。Stowell等人表明通过完全重组的Ccr 4-Not复合物去除poly（A）尾的速率受到底物RNA序列和RNA结合衔接蛋白Mmi 1的影响。
Ccr4-Not is a conserved protein complex that shortens the 3′ poly(A) tails of eukaryotic mRNAs to regulate transcript stability and translation into proteins. RNA-binding proteins are thought to facilitate recruitment of Ccr4-Not to certain mRNAs, but lack of an in-vitro-reconstituted system has slowed progress in understanding the mechanistic details of this specificity. Here, we generate a fully recombinant Ccr4-Not complex that removes poly(A) tails from RNA substrates. The intact complex is more active than the exonucleases alone and has an intrinsic preference for certain RNAs. The RNA-binding protein Mmi1 is highly abundant in preparations of native Ccr4-Not. We demonstrate a high-affinity interaction between recombinant Ccr4-Not and Mmi1. Using in vitro assays, we show that Mmi1 accelerates deadenylation of target RNAs. Together, our results support a model whereby both RNA-binding proteins and the sequence context of mRNAs influence deadenylation rate to regulate gene expression. We purify a fully recombinant S. pombe Ccr4-Not complex The complex has an intrinsic substrate preference, and both Ccr4 and Caf1 are active Ccr4-Not stably interacts with the YTH domain RNA-binding protein Mmi1 Mmi1 accelerates deadenylation of RNAs by Ccr4-Not in a sequence-specific manner Poly(A) tails regulate mRNA stability and translation. Stowell et al. show that the rate of poly(A) tail removal by a fully recombinant Ccr4-Not complex is influenced by both the substrate RNA sequence and an RNA-binding adapter protein, Mmi1.
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影响因子：
13.8
作者：
Bieniossek C;Imasaki T;Takagi Y;Berger I
通讯作者：
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