TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing
TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing
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DOI：
10.1038/nature03868
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发表时间：
2005-08-04
期刊：
影响因子：
64.8
通讯作者：
Shiekhattar, R
中科院分区：
文献类型：
--
作者：
Chendrimada, TP;Gregory, RI;Shiekhattar, R
MicroRNA（miRNA）是由两步处理途径产生的，以产生约22个核苷酸的RNA分子，这些核苷酸约22个核苷酸，这些核苷酸在转录后水平上对靶基因表达产生负调节（1）。微处理器复合物（2-4）将原发性miRNA处理为前体miRNA（前MIRNA）。这些前MIRNA被RNase III迪切尔（5-8）裂解，以产生成熟的miRNA，该miRNA将RNA诱导的沉默复合物（RISC）引导至具有互补序列的Messenger RNA（9）。在这里，我们表明TRBP（人类免疫缺陷病毒反式激活反应RNA结合蛋白（10）），其中包含三个双链的RNA结合结构域，是含DICER的复合物的组成部分。对含TRBP的复合物的生化分析揭示了DICER -TRBP与RISC的催化引擎的Argonaute 2（Ago2）（AGO2）（11,12）的关联。使用FLAG标记的AGO2细胞系分离后，确认了DICER -TRBP和AGO2的物理关联。体外重构测定法证明，将AGO2募集到小型干扰RNA（siRNA）由DICER约束所必需的TRBP。 TRBP的敲低导致DICER不稳定和随之而来的miRNA生物发生丧失。最后，通过外源引入的siRNA减少了dicer -trbp复合物的耗尽，减少了RISC介导的记者基因沉默。这些结果不仅在miRNA加工中，而且作为RISC组装的平台都支持DICER -TRBP复合物的作用。
MicroRNAs ( miRNAs) are generated by a two-step processing pathway to yield RNA molecules of approximately 22 nucleotides that negatively regulate target gene expression at the post-transcriptional level(1). Primary miRNAs are processed to precursor miRNAs (pre-miRNAs) by the Microprocessor complex(2-4). These pre-miRNAs are cleaved by the RNase III Dicer(5-8) to generate mature miRNAs that direct the RNA-induced silencing complex ( RISC) to messenger RNAs with complementary sequence(9). Here we show that TRBP ( the human immunodeficiency virus transactivating response RNA-binding protein(10)), which contains three double-stranded, RNA-binding domains, is an integral component of a Dicer-containing complex. Biochemical analysis of TRBP-containing complexes revealed the association of Dicer - TRBP with Argonaute 2 (Ago2)(11,12), the catalytic engine of RISC. The physical association of Dicer - TRBP and Ago2 was confirmed after the isolation of the ternary complex using Flag-tagged Ago2 cell lines. In vitro reconstitution assays demonstrated that TRBP is required for the recruitment of Ago2 to the small interfering RNA ( siRNA) bound by Dicer. Knockdown of TRBP results in destabilization of Dicer and a consequent loss of miRNA biogenesis. Finally, depletion of the Dicer - TRBP complex via exogenously introduced siRNAs diminished RISC-mediated reporter gene silencing. These results support a role of the Dicer - TRBP complex not only in miRNA processing but also as a platform for RISC assembly.