RNA interference (RNAi) and microRNAs impact eukaryotic genome expression and govern many patho/physiological processes.
We integrate classical biochemistry, genetics, and biophysical approaches to investigate the molecular and regulatory mechanisms of the RNAi and microRNA pathways. We used biochemical fractionation and reconstitution to identify a number of important components of the RNAi/miRNA pathways, including the Drosophila miRNA- (Dicer-1-Loqs) and siRNA- (Dicer-2-R2D2) generating enzymes.
We reconstituted duplex siRNA-initiated RISC activity using recombinant Dicer-2/R2D2 and Ago2. We employed this core reconstitution system to identify C3PO as a novel endoRNase that promotes fly and human RISC activation by degrading the siRNA passenger fragments. We also determined the crystal structure of human C3PO and characterized its catalytic center.
Furthermore, we studied key enzymes, co-factors, and modulators of the miRNA-generating machinery. We found that Sjogren syndrome antigen B (SSB)/La promotes global miRNA expression by stabilizing precursor miRNAs. We discovered that the mitogen activated protein kinase (MAPK)/Erk targets human miRNA machinery to effect mitogenic signaling.
Finally, we have gradually introduced fly genetic techniques into the lab. We generated r2d2 deletion flies and loqs knockout flies by ends-out homologous recombination. We conducted a large-scale EMS mutagenesis genetic screen in Drosophila and identified novel RNAi-defective mutants.
Q. Liu, T. A. Rand, S. Kalidas, F. Du, H. Kim, D.P. Smith, X. Wang, R2D2, a bridge between the initiation and effector steps of the Drosophila RNAi pathway. Science 301:1921-1925. (2003)
(Previewed in Cell 115: 132-133).
The RNA interference (RNAi) pathway is initiated by processing long double-stranded RNA (dsRNA) into small interfering RNA (siRNA). We purified the siRNA-generating enzyme from Drosophila S2 cells and and found that it consists of two stoichiometric subunits: Dicer-2 (DCR-2) and a previously unknown protein that we named R2D2 [two dsRNA-binding domains (R2) and associated with Dicer2 (D2)].
Association with R2D2 does not regulate the siRNA-generating activity of DCR-2. Rather, the DCR-2-R2D2 complex, but not DCR-2 alone, binds and recruits duplex siRNA to Argonaute 2 (Ago2), the “slicer” of RISC, to promote the assembly of the RNA-initiated silencing complex (RISC). Thus, the DCR2-R2D2 complex acts as core component of the RISC loading complex (RLC) and serves as the gateway for siRNA entry into RISC. In an active RISC, a single strand of siRNA (called a guide strand) directs Ago2 to catalyze sequence-specific cleavage of complementary mRNA.
Y. Liu, X. Ye, F. Jiang, C. Liang, D. Chen, J. Peng, L.N. Kinch, N.V. Grishin, and Q. Liu. C3PO, an endoribonuclease that promotes RNAi by facilitating RISC activation. Science,325:750-753 (2009)
(Selected by Faculty of 1000 Biology and Highlighted in Nat Struct Mol Biol. 2009 16:909).
The catalytic engine of RNA interference (RNAi) is the RNA-induced silencing complex (RISC), wherein the endoribonuclease Argonaute and single-stranded small interfering RNA (siRNA) direct target mRNA cleavage.
We reconstituted long double-stranded RNA- and duplex siRNA-initiated RISC activities with the use of recombinant Drosophila Dicer-2, R2D2, and Ago2 proteins. We used this core reconstitution system to purify an RNAi regulator that we term C3PO (component 3 promoter of RISC), a complex of Translin and Trax.
C3PO is a Mg2+-dependent endoribonuclease that promotes RISC activation by removing siRNA passenger strand cleavage products. These studies establish an in vitro RNAi reconstitution system and identify C3PO as a key activator of the core RNAi machinery.