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RNAi Interference by dsRNA Injection into Drosophila Embryos
Published on: April 11, 2011
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定义Drosophila RNAi酶复合物的组装中的基本步骤
Shintaro Iwasaki1, Hiroshi M Sasaki1, Yuriko Sakaguchi2
1Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan.
Nature
|March 31, 2015
概括
伴奏蛋白稳定了siRNA复合体和阿尔戈诺特2 (Ago2) 之间的相互作用,促进了RNA诱导的沉默复合体 (RISC) 的组装. 这一过程对于通过小干扰RNAs (siRNAs) 介导的基因沉默至关重要.
科学领域:
- 分子生物学分子生物学
- RNA干扰 (RNAi) 是一种RNA干扰.
- 蛋白质生物化学 蛋白质生物化学
背景情况:
- 小RNAs,包括siRNAs和miRNAs,通过引导Argonaute (Ago) 蛋白到互补的信使RNAs来调节基因表达.
- RNA诱导沉默复合体 (RISC) 是对RNA沉默至关重要的效应体复合体,其核心是Ago蛋白.
- 在Drosophila Ago2中加载siRNA的精确机制,涉及Dicer-2,R2D2和陪伴机器,仍然不完全理解.
研究的目的:
- 阐明控制RNA诱导沉默复合体 (RISC) 组装的分子机制.
- 为了研究Hsc70/Hsp90陪伴机器在加载小干扰RNA (siRNA) 复杂体到Argonaute 2 (Ago2) 中的作用.
主要方法:
- 在试验室中使用净化组件重组RISC组件:Ago2,Dicer-2,R2D2,HSC70,HSP90,Hop,Droj2和p23.
- 单分子观察以追踪RISC组装中间体的动态.
- 测试用于监测Dicer-2-R2D2-siRNA复合体与Ago2在伴随机械的存在和缺席中的相互作用.
主要成果:
- 由于没有陪伴者,siRNA结合的Dicer-2-R2D2与Ago2的关联是暂时的.
- Hsc70/Hsp90陪伴机器显著延长了Dicer-2-R2D2-siRNA复合物的Ago2.2上的停留时间.
- 这种伴侣介导的稳定性取决于siRNA导向链上的5'-酸盐组的识别.
结论:
- Hsc70/Hsp90陪伴机器在促进有效的siRNA双重加载到Ago2.2中发挥着关键作用.
- 护卫者保持Ago2在有利于从Dicer-2-R2D2复合体生产性siRNA负载的构造中.
- 这项研究提供了对RISC组装的伴侣辅助机制的关键见解,这对于RNA干扰至关重要.
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