Craig Mello looks for more components of RNAi
Today's Journal Club:
Paper: "Functional proteomics reveals the biochemical niche of C. elegans DCR-1 in
multiple small-RNA-mediated pathways."
Duchaine et al..
Cell 124, 343-354, January 27, 2006.
Background: C. elegans has only one Dicer (RNAse III-related enzymes), DCR-1, yet it conducts multiple RNAi pathways: it can process double-stranded RNA to short interfering RNA, it can process pre-microRNA to microRNA, etc. Hypothesis: DCR-1 has interacting proteins that can mediate these multiple functions.
Question: What proteins interact with DCR-1?
Experimental approach: Immunoprecipitate worm fractions with DCR-1 and subject to mass spectrometry analysis to identify the proteins pulled down. The negative control is a worm with a deficiency in dcr-1. Validate the interactions by monitoring RNAi responses in mutants.
Major findings: "Known" interactors were pulled down as candidates, which validates the method. Also, orthologs of "known" interactors in other organisms were pulled down, such as members of the RNA-induced silencing complex (RISC). Some completely new proteins were identified as well: for example, pir-1 mutants do not respond to dsRNA, and no siRNA accumulates in these worms upon exposure to dsRNA.
Implications: Just working on the pathway.
Issues: I don't trust IPs, so it's good they're working on validating their candidates.
How are these interactions regulated? If you feed the worms dsRNA, would you pull down different proteins as RNAi is activated?
Paper: "Functional proteomics reveals the biochemical niche of C. elegans DCR-1 in
multiple small-RNA-mediated pathways."
Duchaine et al..
Cell 124, 343-354, January 27, 2006.
Background: C. elegans has only one Dicer (RNAse III-related enzymes), DCR-1, yet it conducts multiple RNAi pathways: it can process double-stranded RNA to short interfering RNA, it can process pre-microRNA to microRNA, etc. Hypothesis: DCR-1 has interacting proteins that can mediate these multiple functions.
Question: What proteins interact with DCR-1?
Experimental approach: Immunoprecipitate worm fractions with DCR-1 and subject to mass spectrometry analysis to identify the proteins pulled down. The negative control is a worm with a deficiency in dcr-1. Validate the interactions by monitoring RNAi responses in mutants.
Major findings: "Known" interactors were pulled down as candidates, which validates the method. Also, orthologs of "known" interactors in other organisms were pulled down, such as members of the RNA-induced silencing complex (RISC). Some completely new proteins were identified as well: for example, pir-1 mutants do not respond to dsRNA, and no siRNA accumulates in these worms upon exposure to dsRNA.
Implications: Just working on the pathway.
Issues: I don't trust IPs, so it's good they're working on validating their candidates.
How are these interactions regulated? If you feed the worms dsRNA, would you pull down different proteins as RNAi is activated?