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Biochemical analysis of siRNA/miRNA function in human

人类 siRNA/miRNA 功能的生化分析

基本信息

批准号：
7233251
负责人：
THOMAS TUSCHL
金额：
$ 28.45万
依托单位：
ROCKEFELLER UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-06-01 至 2008-05-31
项目状态：
已结题

项目摘要

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.

描述（由申请人提供）：本项目的主要目的是在分子水平上分析人类细胞中由双链RNA（dsRNA）控制的基因表达的调控机制。dsRNA分子由病毒感染或转座子活性产生，或者它们以调控的方式从编码短反向重复序列（microRNA）的细胞基因表达。将dsRNA引入到与细胞基因同源的细胞中，彻底改变了细胞生物学研究，使研究人员能够抑制他们最喜欢的基因进行功能分析，并为基因特异性治疗的新方法打开了大门。该项目的目的是鉴定和表征参与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物如何形成并发挥其功能。1.人siRNA/miRNA-蛋白质复合物的表征。 1.1研究siRNA/miRNA相关Argonaute蛋白成员（eIF 2C家族、HIWI、HIL 1）的作用，并定义它们与RNAi和miRNA介导的基因调控所需的细胞组分的相互作用网络。 1.2从重组蛋白和短双链体或单链siRNA分子重建序列特异性核酸内切酶复合物（RISC）。 2. miRNP/靶mRNA复合物的分离和经受microRNA介导的控制的细胞mRNA的鉴定。