Characterization of the TRBP domain required for Dicer interaction and function in RNA interference
Characterization of the TRBP domain required for Dicer interaction and function in RNA interference
复制标题
DOI：
10.1186/1471-2199-10-38
复制
发表时间：
2009-05-07
影响因子：
--
通讯作者：
Gatignol, Anne
中科院分区：
文献类型：
--
作者：
Daniels, Sylvanne M.;Melendez-Pena, Carlos E.;Gatignol, Anne
背景：Dicer、Ago 2和TRBP是人RNA诱导沉默复合物（RISC）的最小组成部分。虽然Dicer和Ago 2是RNA酶，但TRBP是将小干扰RNA加载到RISC中的双链RNA结合蛋白（dsRBP）。结果：TRBP在Dicer中的结合位点是位于ATP酶和解旋酶结构域之间的165个氨基酸（aa）区域。TRBP的结合位点是一个69个氨基酸的结构域，称为C4，位于TRBP的C-末端。TRBP 1和TRBP 2同种型与Dicer共免疫沉淀，但不与缺乏C4位点的TRBP（TRBP Δ C4）共免疫沉淀。因此，C4结构域是结合Dicer所必需的，而与RNA的存在无关。免疫荧光显示，虽然全长TRBP与Dicer共定位，但TRBP Delta C4不表达TRBP的tarbp 2（-/-）细胞不支持针对EGFP的短发夹或微小RNA介导的RNA干扰（RNAi）。两种TRBP，但不是TRBP Delta C4，都能够挽救RNAi功能。在具有低RNAi活性的人类细胞中，除了TRBP 1或2，而不是TRBP Delta C4，拯救了RNAi function.Conclusion：TRBP和Dicer之间的相互作用位点的映射显示了它们结合所需的独特结构域。由于TRBP Delta C4不与Dicer相互作用或共定位，因此我们建议TRBP和Dicer（均为dsRBP）不通过结合的双链RNA相互作用。TRBP，而不是TRBP Delta C4，在RNAi受损的细胞中拯救RNAi活性，表明Dicer与TRBP的结合对于RNAi功能至关重要。
Background: 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.Results: 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 (TRBPs Delta C4), 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, TRBPs Delta C4 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 TRBPs Delta C4, were able to rescue RNAi function. In human cells with low RNAi activity, addition of TRBP1 or 2, but not TRBPs Delta C4, rescued RNAi function.Conclusion: The mapping of the interaction sites between TRBP and Dicer show unique domains that are required for their binding. Since TRBPs Delta C4 do not interact or colocalize with Dicer, we suggest that TRBP and Dicer, both dsRBPs, do not interact through bound dsRNA. TRBPs, but not TRBPs Delta C4, rescue RNAi activity in RNAi-compromised cells, indicating that the binding of Dicer to TRBP is critical for RNAi function.