Cyclophilin 40 facilitates HSP90-mediated RISC assembly in plants
Cyclophilin 40 facilitates HSP90-mediated RISC assembly in plants
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DOI：
10.1038/emboj.2011.395
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发表时间：
2012-01-18
期刊：
影响因子：
11.4
通讯作者：
Ishikawa, Masayuki
中科院分区：
文献类型：
--
作者：
Iki, Taichiro;Yoshikawa, Manabu;Ishikawa, Masayuki
转录后基因沉默是由包含 AGO 蛋白和单链小 RNA 的 RNA 诱导沉默复合物 (RISC) 介导的。含 AGO1 的植物 RISC 的组装依赖于分子伴侣 HSP90。在这里，我们证明亲环蛋白 40 (CYP40)、蛋白磷酸酶 5 (PP5) 和其他几种具有四三肽重复 (TPR) 结构域的蛋白质在空泡烟草原生质体 (BYL) 提取物中以 HSP90 依赖性方式与 AGO1 结合。有趣的是，CYP40（而非其他 TPR 蛋白）可以与小 RNA 双链体结合的 AGO1 形成复合物。此外，使用 BYL 通过体外翻译合成的 CYP40 独特地促进了小 RNA 双链体与 AGO1 的结合，从而增加了可以切割靶 RNA 的成熟 RISC 的数量。 CYP40 不包含在成熟的 RISC 中，表明这种关联是暂时的。添加 PP5 或亲环蛋白结合药物环孢菌素 A 可阻止内源性 CYP40 与 HSP90-AGO1 复合物的结合并抑制 RISC 组装。这些结果表明，AGO1、HSP90、CYP40 和小 RNA 双链体的复合物是植物中 RISC 组装的关键中间体。 EMBO 杂志 (2012) 31, 267-278。 doi：10.1038/emboj.2011.395； 2011 年 11 月 1 日在线发布
Posttranscriptional gene silencing is mediated by RNA-induced silencing complexes (RISCs) that contain AGO proteins and single-stranded small RNAs. The assembly of plant AGO1-containing RISCs depends on the molecular chaperone HSP90. Here, we demonstrate that cyclophilin 40 (CYP40), protein phosphatase 5 (PP5), and several other proteins with the tetratricopeptide repeat (TPR) domain associates with AGO1 in an HSP90-dependent manner in extracts of evacuolated tobacco protoplasts (BYL). Intriguingly, CYP40, but not the other TPR proteins, could form a complex with small RNA duplex-bound AGO1. Moreover, CYP40 that was synthesized by in-vitro translation using BYL uniquely facilitated binding of small RNA duplexes to AGO1, and as a result, increased the amount of mature RISCs that could cleave target RNAs. CYP40 was not contained in mature RISCs, indicating that the association is transient. Addition of PP5 or cyclophilin-binding drug cyclosporine A prevented the association of endogenous CYP40 with HSP90-AGO1 complex and inhibited RISC assembly. These results suggest that a complex of AGO1, HSP90, CYP40, and a small RNA duplex is a key intermediate of RISC assembly in plants. The EMBO Journal (2012) 31, 267-278. doi:10.1038/emboj.2011.395; Published online 1 November 2011