RNAi Discussion Group
Tuesday, April 11, 2006
Presented by the RNAi Discussion Group
Organizer: Dinshaw Patel, Memorial Sloan-Kettering Cancer Center
The goal of this group is to provide a forum for New York–area scientists engaged in research into the biology, biochemistry, and applications of RNAi silencing to meet regularly to discuss advances in this exciting new field and to promote interinstitutional and interdisciplinary dialogue. Topics of discussion include regulation of RNAi gene expression, mechanistic studies into RNAi action, regulation of gene expression by siRNAs, RNAi in health and disease, and applied uses of technologies related to RNAi.
5:00 pm - 7:30 pm: Presentations
Wei Yang, NIDDK, NIH, "From RNase H to RNAi: Substrate Recognition and Two-metal Ion Catalysis."
Ramin Shiekhattar, The Wistar Institute, "Biogenesis and the Mechanism of Action of microRNAs."
Ian McRae, University of California, Berkeley, "Structural Basis of Double Stranded RNA Processing by Dicer."
Jin-Biao Ma, Memorial Sloan Kettering Cancer Center, "Crystal Structure of siRNA Bound to PAZ-containing Module of Human Dicer and its Implication for Dicer-mediated Recognition of Double-stranded RNA."
"From RNase H to RNAi: Substrate Recognition and Two-metal Ion Catalysis"
RNase H belongs to a nucleotidyl transferase superfamily, which includes transposase, retroviral integrase, Holliday-junction resolvase and RISC nuclease Argonaute. We first determined the crystal structures of RNase H complexed with an RNA/DNA substrate. These structures reveal the mechanism for substrate recognition and two-metal ion-dependent catalysis. RNase H specifically recognizes the A-form RNA strand and the B-form DNA strand. Structure comparisons lead us to predict the catalytic residues of Argonaute and conclude that two-metal ion catalysis is a general feature of the superfamily. Recently, we have determined the crystal structures of RNase H complexed with a catalytic intermediate analog and hydrolysis product. In comparison with the substrate complexes, the two metal ions have altered location and coordination ligands at different steps of reaction. We hypothesize that the movement of two metal ions may assist nucleotidyl transfer reaction by destabilizing the enzyme-substrate complex and facilitating nucleophilic attack and product release.
"Biogenesis and the Mechanism of Action of microRNAs"
RNA interference is implemented through the action of a multiprotein complex termed RNA-induced silencing complex (RISC) programmed through microRNA or siRNA. We describe the biogenesis of microRNA pathway. We will also present evidence supporting a role for the RISC as a pre-assembled stable multiprotein complex composed of Dicer, the double-stranded RNA binding protein TRBP, and Ago2. We demonstrate that this complex could cleave a target RNA using a precursor microRNA (pre-miRNA) hairpin as the source of siRNA. Moreover, the Dicer-containing RISC could distinguish the guide strand of the siRNA from the passenger strand and specifically incorporates only the guide strand into an active RISC. Importantly, while ATP hydrolysis is not required for miRNA processing, assembly of an active RISC, or target RNA cleavage, ATP stimulates target RNA cleavage yielding enhanced RISC activity. These results define the composition of the RISC, and demonstrate that miRNA processing by Dicer and Ago2-mediated target cleavage are coupled.
"Structural Basis of Double Stranded RNA Processing by Dicer"
The specialized ribonuclease Dicer initiates RNA interference (RNAi) by cleaving double stranded RNA (dsRNA) substrates into small fragments about 25 nucleotides in length. In the crystal structure of an intact Dicer enzyme, the PAZ domain, a dsRNA-end binding module, is separated from the two catalytic RNase III domains b