CCR4-NOT Deadenylates mRNA Associated with RNA-Induced Silencing Complexes in Human Cells
CCR4-NOT Deadenylates mRNA Associated with RNA-Induced Silencing Complexes in Human Cells
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CCR4-NOT 使与人类细胞中 RNA 诱导的沉默复合物相关的 mRNA 脱腺苷酸化
DOI：
10.1128/mcb.01481-09
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发表时间：
2010-03-15
影响因子：
5.3
通讯作者：
Belasco, Joel G.
中科院分区：
文献类型：
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作者：
Piao, Xianghua;Zhang, Xue;Belasco, Joel G.
摘要微小RNA（miRNAs）通过抑制翻译和加速去腺苷酸化来抑制转录后基因表达，从而引发mRNA的快速降解。它们的调节作用是由RNA诱导沉默复合物（RISC）的蛋白组分介导的，RISC将miRNA和siRNA递送到其mRNA靶点。在这里，我们提出的证据表明，CCR 4-NOT是去腺苷酸酶，从人类细胞中的miRNA不稳定的信息中去除poly（A）。过量产生该去腺苷酶的催化亚基（CCR 4或CAF 1/POP 2）中的任一种的突变失活形式显著阻碍由部分互补的miRNA靶向的mRNA的去腺苷化和衰变。相同的去腺苷酶启动“脱靶”mRNA的降解，所述mRNA被通过转染引入的不完全互补的siRNA结合。无活性的CAF 1或POP 2（相对于无活性的CCR 4）的抑制作用更大，表明CCR 4-NOT的催化亚基在miRNA或小干扰RNA（siRNA）介导的去腺苷化中起主导作用。mi/siRNA和CCR 4-NOT的这些作用可以通过直接将RISC拴系到mRNA而不需要小RNA的指导来完全再现，这表明RISC加速去腺苷化的能力不依赖于RNA碱基配对。尽管CCR 4-NOT对mi/siRNA介导的去腺苷化很重要，但它似乎与RISC没有显著关联，这是通过CAF 1和POP 2未能与RISC的Ago或TNRC 6亚基可检测地共免疫沉淀来判断的，这一发现与去腺苷酶募集作为RISC加速poly（A）去除的机制不一致。
ABSTRACT MicroRNAs (miRNAs) repress gene expression posttranscriptionally by inhibiting translation and by expediting deadenylation so as to trigger rapid mRNA decay. Their regulatory influence is mediated by the protein components of the RNA-induced silencing complex (RISC), which deliver miRNAs and siRNAs to their mRNA targets. Here, we present evidence that CCR4-NOT is the deadenylase that removes poly(A) from messages destabilized by miRNAs in human cells. Overproducing a mutationally inactivated form of either of the catalytic subunits of this deadenylase (CCR4 or CAF1/POP2) significantly impedes the deadenylation and decay of mRNA targeted by a partially complementary miRNA. The same deadenylase initiates the degradation of “off-target” mRNAs that are bound by an imperfectly complementary siRNA introduced by transfection. The greater inhibitory effect of inactive CAF1 or POP2 (versus inactive CCR4) suggests a predominant role for this catalytic subunit of CCR4-NOT in miRNA- or small interfering RNA (siRNA)-mediated deadenylation. These effects of mi/siRNAs and CCR4-NOT can be fully reproduced by directly tethering RISC to mRNA without the guidance of a small RNA, indicating that the ability of RISC to accelerate deadenylation is independent of RNA base pairing. Despite its importance for mi/siRNA-mediated deadenylation, CCR4-NOT appears not to associate significantly with RISC, as judged by the failure of CAF1 and POP2 to coimmunoprecipitate detectably with either the Ago or TNRC6 subunit of RISC, a finding at odds with deadenylase recruitment as the mechanism by which RISC accelerates poly(A) removal.