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.