RNAi Discussion Group
Thursday, March 17, 2005
Presented by the RNAi Discussion Group
Organizer: Gregory Hannon, Cold Spring Harbor Laboratory
The RNAi Discussion Group has been organized under the leadership of Thomas Tuschl, The Rockefeller University; Gregory Hannon, Cold Spring Harbor Laboratories; and Timothy Bestor, Columbia University.
Danesh Moazed, "RNAi-Mediated Assembly of Epigenetic Chromatin Domains."
The RNA interference pathway is required for transcriptional gene silencing in many organisms. Our laboratory has purified a ribonucleoprotein complex, termed RITS (RNA-induced transcriptional silencing), which physically links the RNAi pathway to heterochromatin assembly in fission yeast. RITS contains the conserved siRNA-binding protein, Ago1, a chromodomain histone binding protein, Chp1, and a novel protein, Tas3. Moreover, RITS contains siRNAs that match the sequence of fission yeast centromeric repeats, which are assembled into heterochromatin, and siRNAs are required for the association of RITS with its target DNA regions. These results suggest that RITS uses siRNAs as guides to initiate heterochromatin assembly at specific chromosome regions. More recently, we have found that RITS associates with another RNAi complex that contains the fission yeast RNA-directed RNA polymerase, Rdp1, termed RDRC (RNA-directed RNA polymerase complex). The association of RITS with RDRC requires the Dicer ribonuclease and the Clr4 histone H3-lysine9 methyltransferase, suggesting that both siRNA and chromatin localization contribute to this interaction. In addition to Rdp1, RDRC contains a conserved RNA helicase and a protein that belongs to the polyA polymerase family of enzymes. We have shown that RDRC has RNA-directed RNA polymerase activity and that this activity is required for heterochromatin assembly at centromeres. Finally, components of RITS and RDRC associate with noncoding centromeric RNAs in an siRNA-directed manner. Our findings suggest that nascent noncoding RNAs provide a platform for the assembly of RNAi complexes at specific chromosome regions.
Fabiola Rivas, "Purified Argonaute2 and an siRNA Form a Recombinant Human RNA-Induced Silencing Complex."
Genetic, biochemical, and structural studies have implicated Argonaute proteins as the catalytic core of the RNAi effector complex, recombinant human RNA-induced silencing complex (RISC). Here we show that recombinant human Argonaute2 can combine with an siRNA to form a minimal RISC that accurately cleaves substrate RNAs. The recombinant RISC shows many of the properties of RISC purified from human or Drosophila cells but also surprising features. It shows no stimulation by ATP, suggesting that factors promoting product release are missing from the recombinant enzyme. The active site constitutes a unique “DDH” motif. The RISC reconstitution system reveals that the siRNA 5′ phosphate is important for the stability and the fidelity of the complex but is not essential for the creation of an active enzyme. These studies demonstrate that Argonaute proteins catalyze mRNA cleavage within the RISC and provide a source of recombinant enzyme for detailed biochemical studies of the RNAi effector complex.
Antonio Giraldez, "MicroRNAs Regulate Brain Morphogenesis and Other Aspects of Zebrafish Embryogenesis."
MicroRNAs (miRNAs) are small RNAs that regulate gene expression posttranscriptionally. To block all miRNA f
This meeting has been cancelled due to speaker cancellation. The next RNAi discussion group meeting will be held on Thursday, April 21, 2005 and will include talks by Victor Ambros (Dartmouth), Antonio Giraldez (NYU) and others.