RNA Silencing Complex Assembly and Function

描述（由申请人提供）：来自不同真核生物的细胞沉默同源基因表达以响应称为sirna（短干扰rna）和miRNAs （microRNAs）的小rna。这些RNA沉默机制可以控制细胞基因的表达，指定特定染色质结构域的功能，并保护细胞免受病毒感染和转座子入侵。RNA沉默的一种形式是RNA干扰（RNAi），它指导与siRNA具有相同序列的mRNA转录物的特异性降解。转录本破坏是由rna诱导的沉默复合体（RISC）执行的。本研究的目的是利用体外对RNAi有活性的黑腹果蝇胚胎裂解物，确定RISC组装和功能的生化途径。果蝇的RISCs大小不同，但它们之间的关系尚不清楚，而且它们是如何组装的几乎一无所知。此外，sirna导向的内切酶的身份仍然未知。我们已经开发了一种新的天然凝胶电泳测定，我们可以用来监测在放射性标记的siRNA上形成的蛋白质复合物。我们已经使用该方法鉴定了至少三个复合物，其中两个（称为R1和R2）是RISC组装途径的中间产物。另一种复合体R3是一种非常大的（80S）且可能与核糖体相关的RISC形式，可以特异性识别和切割目标mrna。本项目旨在通过三种方式进一步定义RNAi通路：1)通过表征中间复合物并确定其组装成高阶沉默复合物的要求；2)通过纯化R3复合体来鉴定存在于这种新型RISC中的蛋白质；3)通过位点特异性光交联鉴定在RISC组装、激活和功能过程中与目标mRNA的功能不同区域（包括切割位点）接触的RISC组分。

DESCRIPTION (provided by applicant): Cells from diverse eukaryotes silence cognate gene expression in response to small RNAs called siRNAs (short interfering RNAs) and miRNAs (microRNAs). These RNA silencing mechanisms can govern the expression of cellular genes, specify the functions of specific chromatin domains, and protect cells from viral infection and transposon invasion. One form of RNA silencing, RNA interference (RNAi), directs the specific degradation of mRNA transcripts that share sequence identity with an siRNA. Transcript destruction is executed by the RNA-induced silencing complex (RISC). The goal of the proposed research is to determine the biochemical pathway of RISC assembly and function, using Drosophila melanogaster embryo lysates that are active for RNAi in vitro. Drosophila RISCs of differing sizes have been reported, but the relationships between them are unclear, and almost nothing is known about how they assemble. Furthermore, the identity of the siRNA-directed endonuclease remains unknown. We have developed a novel native gel electrophoresis assay that we can use to monitor protein complexes that form on radiolabeled siRNA. We have used this assay to identify at least three complexes, two of which (called R1 and R2) are intermediates in a RISC assembly pathway. The other complex, R3, is a very large (80S) and potentially ribosome-associated form of RISC that can specifically recognize and cleave targeted mRNAs. This project aims to further define the RNAi pathway in three ways: 1) By characterizing the intermediate complexes and determining the requirements for their assembly into higher-order silencing complexes; 2) By purifying the R3 complex to identify proteins present within this novel form of RISC; and 3) By employing site-specific photocrosslinking to identify RISC components that contact functionally distinct regions of the target mRNA (including the cleavage site) during RISC assembly, activation and function. Six years after the initial discovery of RNA silencing, it is clear that these pathways are vital to many facets of biology. Moreover, RNAi has become an indispensable experimental tool, and has the potential to become a very powerful therapeutic technique. Accordingly, a deeper understanding of RNA silencing pathways will accelerate many aspects of biomedical research and disease treatment.