Daniels Sylvanne M, Melendez-Peña Carlos E, Scarborough Robert J, Daher Aïcha, Christensen Helen S, El Far Mohamed, Purcell Damian F J, Lainé Sébastien, Gatignol Anne
Virus-Cell Interactions Laboratory, Lady Davis Institute for Medical Research, Montréal, Québec, Canada.
BMC Mol Biol. 2009 May 7;10:38. doi: 10.1186/1471-2199-10-38.
Dicer, Ago2 and TRBP are the minimum components of the human RNA-induced silencing complex (RISC). While Dicer and Ago2 are RNases, TRBP is the double-stranded RNA binding protein (dsRBP) that loads small interfering RNA into the RISC. TRBP binds directly to Dicer through its C-terminal domain.
We show that the TRBP binding site in Dicer is a 165 amino acid (aa) region located between the ATPase and the helicase domains. The binding site in TRBP is a 69 aa domain, called C4, located at the C-terminal end of TRBP. The TRBP1 and TRBP2 isoforms, but not TRBPs lacking the C4 site (TRBPsDeltaC4), co-immunoprecipitated with Dicer. The C4 domain is therefore necessary to bind Dicer, irrespective of the presence of RNA. Immunofluorescence shows that while full-length TRBPs colocalize with Dicer, TRBPsDeltaC4 do not. tarbp2-/- cells, which do not express TRBP, do not support RNA interference (RNAi) mediated by short hairpin or micro RNAs against EGFP. Both TRBPs, but not TRBPsDeltaC4, were able to rescue RNAi function. In human cells with low RNAi activity, addition of TRBP1 or 2, but not TRBPsDeltaC4, rescued RNAi function.
The mapping of the interaction sites between TRBP and Dicer show unique domains that are required for their binding. Since TRBPsDeltaC4 do not interact or colocalize with Dicer, we suggest that TRBP and Dicer, both dsRBPs, do not interact through bound dsRNA. TRBPs, but not TRBPsDeltaC4, rescue RNAi activity in RNAi-compromised cells, indicating that the binding of Dicer to TRBP is critical for RNAi function.
Dicer、Ago2和TRBP是人类RNA诱导沉默复合体（RISC）的最小组成部分。Dicer和Ago2是核糖核酸酶，而TRBP是双链RNA结合蛋白（dsRBP），它将小干扰RNA加载到RISC中。TRBP通过其C末端结构域直接与Dicer结合。
我们发现Dicer中的TRBP结合位点是位于ATP酶和螺旋酶结构域之间的一个165个氨基酸（aa）的区域。TRBP中的结合位点是一个69 aa的结构域，称为C4，位于TRBP的C末端。TRBP1和TRBP2亚型，但缺乏C4位点的TRBP（TRBPsDeltaC4）不能与Dicer进行共免疫沉淀。因此，无论RNA是否存在，C4结构域对于结合Dicer都是必需的。免疫荧光显示，全长TRBP与Dicer共定位，而TRBPsDeltaC4则不然。不表达TRBP的tarbp2-/-细胞不支持短发夹或微小RNA介导的针对EGFP的RNA干扰（RNAi）。两种TRBP都能挽救RNAi功能，但TRBPsDeltaC4不能。在RNAi活性较低的人类细胞中，添加TRBP1或2能挽救RNAi功能，但TRBPsDeltaC4不能。
TRBP与Dicer之间相互作用位点的定位显示了它们结合所需的独特结构域。由于TRBPsDeltaC4不与Dicer相互作用或共定位，我们认为TRBP和Dicer这两种dsRBP不是通过结合的dsRNA相互作用的。TRBP能挽救RNAi受损细胞中的RNAi活性，但TRBPsDeltaC4不能，这表明Dicer与TRBP的结合对RNAi功能至关重要。
