Biochemical analysis of siRNA/miRNA function in human

描述(申请人提供)：这个项目的广泛目标是在分子水平上分析人类细胞中双链RNA(DsRNA)控制的基因表达的调控机制。DsRNA分子要么由病毒感染或转座子活动产生，要么由编码短反向重复序列(MicroRNAs)的细胞基因以受调控的方式表达。将dsRNA引入与细胞基因同源的细胞中，使细胞生物学研究发生了革命性的变化，使研究人员能够抑制他们最喜欢的基因进行功能分析，并打开了新的基因特异性治疗方法的大门。本项目的目的是确定和表征参与RNA沉默的蛋白质组分，并在分子水平上了解沉默活性核糖核蛋白复合体是如何形成并发挥其功能的。1.人siRNA/miRNA-蛋白质复合体的鉴定 1.1研究siRNA/miRNA相关的ArgAerte蛋白成员(elF2C家族、HIWI、Hill)的作用，并确定它们与RNAi和miRNA介导的基因调控所需的细胞组件的相互作用网络。 1.2重组蛋白质和短双链或单链siRNA分子重组序列特异性核酸内切酶复合体(RISC)。 2.miRNP/靶mRNA复合体的分离及受microRNA调控的细胞mRNAs的鉴定

DESCRIPTION (provided by applicant): The broad objective of this project is to analyze at the molecular level the regulatory mechanisms of gene expression controlled by double-stranded RNA (dsRNA) in human cells. Molecules of dsRNA are either produced by viral infection or transposon activity, or they are expressed in a regulated manner from cellular genes that encode short inverted repeat sequences (microRNAs). Introduction of dsRNA into cells cognate to cellular genes has revolutionized cell biological studies enabling investigators to suppress their favorite gene for functional analysis and has opened the doors to new ways of gene-specific therapy. The aim of this project is to identify and characterize the protein components involved in RNA silencing and to understand at molecular level how silencing-active ribonucleoprotein complexes are formed and exert their function. 1. Characterization of the human siRNA/miRNA-protein complexes. 1.1 Investigate the role of siRNA/miRNA-associated Argonaute protein members (elF2C family, HIWI, HILl) and define their interaction network with cellular components required for RNAi and miRNA-mediated gene regulation. 1.2 Reconstitute the sequence-specific endonuclease complex (RISC) from recombinant proteins and short duplex or single-stranded siRNA molecules. 2. Isolation of miRNP/target mRNA complexes and identification of the cellular mRNAs subjected to microRNA-mediated control.