RNA. 2013 Nov;19(11):1583-93. doi: 10.1261/rna.040790.113. Epub 2013 Sep 18.
Argonaute2 (Ago2) protein and associated microRNAs (miRNAs) or small interfering RNAs (siRNAs) form the RNA-induced silencing complex (RISC) for target messenger RNA cleavage and post-transcriptional gene silencing. Although Ago2 is essential for RISC activity, the mechanism of RISC assembly is not well understood, and factors controlling Ago2 protein expression are largely unknown. A role for the Hsc70/Hsp90 chaperone complex in loading small RNA duplexes into the RISC has been demonstrated in cell extracts, and unloaded Ago2 is unstable and degraded by the lysosome in mammalian cells. Here we identify the co-chaperones Fkbp4 and Fkbp5 as Ago2-associated proteins in mouse embryonic stem cells. Pharmacological inhibition of this interaction using FK506 or siRNA-mediated Fkbp4/5 depletion leads to decreased Ago2 protein levels. We find FK506 treatment inhibits, whereas Fkbp4/5 overexpression promotes, miRNA-mediated stabilization of Ago2 expression. Simultaneous treatment with a lysosome inhibitor revealed the accumulation of unloaded Ago2 complexes in FK506-treated cells. We find that, consistent with unloaded miRNAs being unstable, FK506 treatment also affects miRNA abundance, particularly nascent miRNAs. Our results support a role for Fkbp4/5 in RISC assembly.
Argonaute2 (Ago2) 蛋白及其相关 microRNAs (miRNAs) 或 small interfering RNAs (siRNAs) 形成 RNA 诱导沉默复合物 (RISC)，用于靶向信使 RNA 的切割和转录后基因沉默。尽管 Ago2 对于 RISC 活性是必不可少的，但 RISC 组装的机制还不太清楚，控制 Ago2 蛋白表达的因素在很大程度上也是未知的。在细胞提取物中已经证明了 Hsc70/Hsp90 伴侣复合物在将小 RNA 双链体加载到 RISC 中的作用，并且未加载的 Ago2 在哺乳动物细胞中不稳定，并被溶酶体降解。在这里，我们在小鼠胚胎干细胞中鉴定了伴侣蛋白 Fkbp4 和 Fkbp5 作为 Ago2 的相关蛋白。使用 FK506 或 siRNA 介导的 Fkbp4/5 耗竭抑制这种相互作用会导致 Ago2 蛋白水平降低。我们发现 FK506 处理抑制，而 Fkbp4/5 过表达促进 miRNA 介导的 Ago2 表达稳定。同时用溶酶体抑制剂处理显示 FK506 处理细胞中未加载的 Ago2 复合物的积累。我们发现，与未加载的 miRNAs 不稳定一致，FK506 处理还会影响 miRNA 的丰度，特别是新生 miRNAs。我们的结果支持 Fkbp4/5 在 RISC 组装中的作用。
