| 研究実績の概要 |
The catalytic engine of RNA interference (RNAi) is the RNA-induced silencing complex (RISC). We identified TAF11 as the missing component, in addition to Dicer-2 and R2D2, of the Drosophila RISC loading complex (RLC) by forward genetic screening. We reconstituted the RLC using recombinant Dicer-2, R2D2 and TAF11 proteins. We showed that TAF11 could promote assembly of RLC by facilitating the formation of Dicer-2/R2D2 heterotetramer. We generated a series of mutant TAF11 by site-directed mutagenesis and showed that several of these mutants were defective for the RLC formation by native gel-shift assays. Accordingly, we found that some of these mutant TAF11 proteins failed to co-IP co-localize with the Dicer-2/R2D2 complex following co-transfection into S2 cells. On top of this, we recently identified histone H1-like protein, HP1BP3, as a chromatin retention factor that promotes co-transcriptional processing of primary microRNAs in mammalian cells.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
We have an excellent research team and received help from outstanding collaborators.
1. We collaborated with Dr. Mikiko Siomi at University of Tokyo, who is a world’s leader in small RNA research, to conduct co-localization studies of GFP-TAF11 and Dicer-2/R2D2, using excellent anti-Dicer-2 and anti-R2D2 monoclonal antibodies that were previously generated in the Siomi lab.
2. Our lab has strong expertise in classical biochemistry. We are good at generating highly purified recombinant Dicer-2/R2D2 and TAF11 proteins using insect cell-expression system. We developed all of the in vitro assays for analysis of RLC and RISC assembly.
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| 今後の研究の推進方策 |
1. To map the molecular interfaces among Dicer-2, R2D2, TAF11 and siRNA within the RLC.
2. To compare the activities of wild-type and mutant TAF11 proteins in the in vitro RISC reconstitution system that we’ve previously established.
3. To generate taf11 knock-in mutant flies by CRISPR/Cas9 technology.
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