RISC is a 5′ phosphomonoester-producing RNA endonuclease
RISC is a 5′ phosphomonoester-producing RNA endonuclease
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DOI：
10.1101/gad.1187904
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发表时间：
2004-05-01
影响因子：
10.5
通讯作者：
Tuschl, T
中科院分区：
文献类型：
--
作者：
Martinez, J;Tuschl, T
RNA干扰过程中的基因沉默是由核糖核蛋白复合物介导的，称为RNA诱导沉默复合物（RISC）。在这里，我们描述了使用亲和纯化的最小RISC从人类细胞的靶RNA切割的分子机制。裂解通过水解和3 '-羟基和5'-磷酸末端的释放进行。在切割位点处的2 '-羟基被2'-脱氧取代没有显著影响，表明产物释放和/或构象转变而不是化学步骤是限速的。在切割位点被2 '-O-甲基取代基本上减少了切割，这可能是由于空间干扰。靶RNA的突变分析显示，siRNA的5'或3'末端的错配几乎没有影响，并且短至15个核苷酸的底物RNA被RISC切割。
Gene silencing in the process of RNA interference is mediated by a ribonucleoprotein complex referred to as RNA-induced silencing complex (RISC). Here we describe the molecular mechanism of target RNA cleavage using affinity-purified minimal RISC from human cells. Cleavage proceeds via hydrolysis and the release of a 3'-hydroxyl and a 5'-phosphate terminus. Substitution of the 2'-hydroxyl group at the cleavage site by 2'-deoxy had no significant effect, suggesting that product release and/or a conformational transition rather than a chemical step is rate-limiting. Substitution by 2'-O-methyl at the cleavage site substantially reduced cleavage, which is presumably due to steric interference. Mutational analysis of the target RNA revealed that mismatches across from the 5' or the 3' end of the siRNA had little effect and that substrate RNAs as short as 15 nucleotides were cleaved by RISC.