The overall goal of this research proposal is to understand the regulatory mechanisms of RNA interference (RNAi). RNAi is a post-transcriptional gene-silencing mechanism mediated by microRNA (miRNA) and small interfering RNA (siRNA). Although a number of RNAi components have been identified, their specific functions in RNAi remain unclear. To date, only three core components are biochemically validated. Dicer processes long dsRNA and pre-miRNA to siRNA and miRNA. Argonaute (Ago), guided by a siRNA or miRNA, directs sequence-specific cleavage and translational repression cognate mRNA. DsRNA-binding protein (dsRBP), such as R2D2 in flies, facilitates siRNA transfer from Dicer to Ago to form the effector RNA-induced silencing (RISC) complex. Biochemical fractionation and reconstitution is a powerful and essential approach to understand the in-depth molecular mechanisms of RNAi. In the current study, we propose to reconstitute the holo-RISC activities by using recombinant Dicer/dsRBP/Ago proteins. Our reconstitution system will place us in a unique position to address several fundamental and long standing questions in the field: What factors constitute holo-RISC? How is RISC assembled? How is RNAi regulated? To achieve our objectives, we have devised three specific avenues of research.
In Aim 1, we have reconstituted Drosophila dsRNA-initiated RISC activity using purified recombinant Dcr- 2/R2D2/Ago2 proteins. This reconstitution system will allow us to investigate the in-depth biochemical mechanisms of RISC assembly.
In Aim 2, we will employ a combinatory genetic and biochemical approach to identify novel regulators of Drosophila RNAi and study the biochemical mechanisms of RNAi regulation.
In Aim 3, we will reconstitute human holo-RISC activity using recombinant Dicer/dsRBP/Ago2 proteins and investigate regulation of human RNAi by phosphorylation of dsRBP. The RNAi and related pathways emerge as a fundamental and global mechanism to control genome activities. Studies of the regulatory mechanisms of RNAi will connect the field to a wider biological context and provide the biochemical basis for normal and pathological regulations of small regulatory RNAs in biology and disease. On the other hand, the mechanistic understanding of RNAi will facilitate rational design of new and improved gene-silencing technologies. Applications of RNAi silencing technologies will have a significant impact on a broad spectrum of biomedical sciences, ranging from understanding the basic biology, uncovering the molecular basis of human disease, to developing novel therapeutics.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM084010-02
Application #
7603081
Study Section
Molecular Genetics B Study Section (MGB)
Program Officer
Bender, Michael T
Project Start
2008-05-01
Project End
2012-03-31
Budget Start
2009-04-01
Budget End
2010-03-31
Support Year
2
Fiscal Year
2009
Total Cost
$282,600
Indirect Cost
Name
University of Texas Sw Medical Center Dallas
Department
Biochemistry
Type
Schools of Medicine
DUNS #
800771545
City
Dallas
State
TX
Country
United States
Zip Code
75390
Li, Liande; Gu, Weifeng; Liang, Chunyang et al. (2012) The translin-TRAX complex (C3PO) is a ribonuclease in tRNA processing. Nat Struct Mol Biol 19:824-30
Li, Yujing; Lin, Li; Li, Zigang et al. (2012) Iron homeostasis regulates the activity of the microRNA pathway through poly(C)-binding protein 2. Cell Metab 15:895-904
Liu, Ying; Liu, Qinghua (2011) ATM signals miRNA biogenesis through KSRP. Mol Cell 41:367-8
Ye, Xuecheng; Huang, Nian; Liu, Ying et al. (2011) Structure of C3PO and mechanism of human RISC activation. Nat Struct Mol Biol 18:650-7
Liu, Ying; Tan, Huiling; Tian, Hui et al. (2011) Autoantigen La promotes efficient RNAi, antiviral response, and transposon silencing by facilitating multiple-turnover RISC catalysis. Mol Cell 44:502-8
Okamura, Katsutomo; Robine, Nicolas; Liu, Ying et al. (2011) R2D2 organizes small regulatory RNA pathways in Drosophila. Mol Cell Biol 31:884-96
Paroo, Zain; Ye, Xuecheng; Chen, She et al. (2009) Phosphorylation of the human microRNA-generating complex mediates MAPK/Erk signaling. Cell 139:112-22
Liu, Ying; Ye, Xuecheng; Jiang, Feng et al. (2009) C3PO, an endoribonuclease that promotes RNAi by facilitating RISC activation. Science 325:750-3
Ye, Xuecheng; Liu, Qinghua (2008) Expression, purification, and analysis of recombinant Drosophila Dicer-1 and Dicer-2 enzymes. Methods Mol Biol 442:11-27