Alternative RISC assembly: Binding and repression of microRNA-mRNA duplexes by human Ago proteins
Alternative RISC assembly: Binding and repression of microRNA-mRNA duplexes by human Ago proteins
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DOI：
10.1261/rna.035675.112
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发表时间：
2012-11-01
期刊：
影响因子：
4.5
通讯作者：
Novina, Carl D.
中科院分区：
文献类型：
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作者：
Janas, Maja M.;Wang, Bingbing;Novina, Carl D.
microRNAs（miRNAs）是一种小的非编码RNA，在转录后调节大多数人类mRNA的蛋白质输出。与所有miRNA都与Argonaute（Ago）蛋白相关的共识观点相反，我们确定了HeLa细胞中miRNA的表达量是Agos的13倍，并且miRNA与mRNA的结合量是Agos的7倍，这意味着存在不与Agos化学计量结合的miRNA-mRNA双链体。我们发现，所有四个人Agos可以抑制miRNA-mRNA双链体，但只有Ago 2可以在体外切割小干扰RNA-mRNA双链体。我们使用荧光寿命成像显微镜观察Ago与活细胞中miRNA-mRNA双链体的直接结合。与Agos结合miRNA双链体的共识观点相反，这些数据表明Agos可以结合并抑制miRNA-mRNA双链体，并支持Ago在翻译抑制中的催化功能模型。
MicroRNAs (miRNAs) are small noncoding RNAs that post-transcriptionally regulate protein output from the majority of human mRNAs. In contrast to the consensus view that all miRNAs are associated with Argonaute (Ago) proteins, we determine that miRNAs are expressed in a 13-fold excess relative to Agos in HeLa cells and that miRNAs are bound to mRNAs in a sevenfold excess relative to Agos, implying the existence of miRNA-mRNA duplexes not stoichiometrically bound by Agos. We show that all four human Agos can repress miRNA-mRNA duplexes, but only Ago2 can cleave small interfering RNA-mRNA duplexes in vitro. We visualize direct Ago binding to miRNA-mRNA duplexes in live cells using fluorescence lifetime imaging microscopy. In contrast to the consensus view that Agos bind miRNA duplexes, these data demonstrate that Agos can bind and repress miRNA-mRNA duplexes and support a model of catalytic Ago function in translational repression.