2016 Fiscal Year Annual Research Report
RNA干渉におけるRISC形成への核内転写因子の関与
| Project/Area Number |
16H04740
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| Research Institution | University of Tsukuba |
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Principal Investigator |
Liu Qinghua 筑波大学, 国際統合睡眠医科学研究機構, 教授 (90723792)
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| Project Period (FY) |
2016-04-01 – 2019-03-31
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| Keywords | 核酸 / RNA / RISC |
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| Outline of Annual Research Achievements |
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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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
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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| Strategy for Future Research Activity |
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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[Journal Article] HP1BP3, a chromatin retention factor for co-transcriptional microRNA processing2016
Author(s)
H. Liu#, C. Liang#, R.K. Kollipara, M. Matsui, X. Ke, B-C. Jeong, Z. Wang, K.S. Yoo, G.P. Yadav, L.N. Kinch, N.V. Grishin, Y. Nam, D.R. Corey, R. Kittler, Q.Liu.
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Journal Title
Mol Cell
Volume: 63
Pages: 420-432
DOI
Peer Reviewed / Int'l Joint Research / Acknowledgement Compliant
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