Liang Chunyang, Wang Yibing, Murota Yukiko, Liu Xiang, Smith Dean, Siomi Mikiko C, Liu Qinghua
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
Department of Biological Sciences, University of Tokyo, Tokyo 113-0032, Japan.
Mol Cell. 2015 Sep 3;59(5):807-18. doi: 10.1016/j.molcel.2015.07.006. Epub 2015 Aug 6.
Assembly of the RNA-induced silencing complex (RISC) requires formation of the RISC loading complex (RLC), which contains the Dicer-2 (Dcr-2)-R2D2 complex and recruits duplex siRNA to Ago2 in Drosophila melanogaster. However, the precise composition and action mechanism of Drosophila RLC remain unclear. Here we identified the missing factor of RLC as TATA-binding protein-associated factor 11 (TAF11) by genetic screen. Although it is an annotated nuclear transcription factor, we found that TAF11 also associated with Dcr-2/R2D2 and localized to cytoplasmic D2 bodies. Consistent with defective RLC assembly in taf11(-/-) ovary extract, we reconstituted the RLC in vitro using the recombinant Dcr-2-R2D2 complex, TAF11, and duplex siRNA. Furthermore, we showed that TAF11 tetramer facilitates Dcr-2-R2D2 tetramerization to enhance siRNA binding and RISC loading activities. Together, our genetic and biochemical studies define the molecular nature of the Drosophila RLC and elucidate a cytoplasmic function of TAF11 in organizing RLC assembly to enhance RNAi efficiency.
RNA诱导沉默复合体（RISC）的组装需要形成RISC装载复合体（RLC），该复合体包含Dicer-2（Dcr-2）-R2D2复合体，并将双链小干扰RNA（siRNA）招募至果蝇的AGO2蛋白上。然而，果蝇RLC的确切组成和作用机制仍不清楚。在此，我们通过遗传筛选确定了RLC缺失的因子为TATA结合蛋白相关因子11（TAF11）。尽管它是一种已注释的核转录因子，但我们发现TAF11也与Dcr-2/R2D2相关，并定位于细胞质中的D2小体。与taf11（-/-）卵巢提取物中RLC组装缺陷一致，我们使用重组Dcr-2-R2D2复合体、TAF11和双链siRNA在体外重建了RLC。此外，我们表明TAF11四聚体促进Dcr-2-R2D2四聚化，以增强siRNA结合和RISC装载活性。总之，我们的遗传和生化研究确定了果蝇RLC的分子性质，并阐明了TAF11在组织RLC组装以提高RNA干扰效率方面的细胞质功能。
