Characterization of TRBP-containing complexes

描述（由申请人提供）：C.线虫需要控制幼虫发育和神经元模式的时间。这些基因中的绝大多数在哺乳动物中是保守的，最近的实验证明了它们在造血分化和癌症的调节中的作用。我们对microRNA途径的生物化学表征揭示了RNase III酶Dicer与致癌蛋白TRBP的关联。Dicer是负责产生成熟microRNA和将外源性双链RNA加工成短干扰RNA（siRNA）的酶。含TRBP复合物的生化分析不仅证实了TRBP和Dicer的缔合，而且还发现了Argonaute 2的存在，Argonaute 2是RNA诱导沉默复合物的催化引擎，作为Dicer/TRBP复合物的稳定组分。我们假设这三种蛋白质形成了负责处理和执行RNAi反应的复合物的核心。我们还发现了证据表明，60 S核糖体亚基与TRBP结合形成一种复合物，参与介导microRNA介导的翻译抑制。目的1明确Dicer/TRBP复合物在siRNA/miRNA形成中的功能，并开始研究单个蛋白及其结构域在siRNA/miRNA加工活性调控中的作用。目的2描述了Dicer/TRBP复合物与含有Ago 2亚基的效应复合物的关联的详细功能分析，并描绘了单个蛋白在转录后基因沉默功能中的作用。目的3将工作扩展到分析含有TRBP的大复合物，该复合物与60 S核糖体亚基和参与翻译抑制的人Armitage蛋白相关。

DESCRIPTION (provided by applicant): The microRNA-group of genes of C. elegans is required to control the timing of larval development and neuronal patterning. A predominant number of these genes are conserved in mammals and recent experiments demonstrated a role for them in modulation of hematopoietic differentiation and cancer. Our biochemical characterization of microRNA pathway revealed the association of the RNase III enzyme Dicer with the oncogenic protein TRBP. Dicer is the enzyme responsible for the generation of mature microRNA and processing of exogenous double-stranded RNA to short interfering RNAs (siRNAs). Biochemical analysis of TRBP-containing complexes not only confirmed the association of TRBP and Dicer but also uncovered the presence of Argonaute 2, the catalytic engine of RNA-induced silencing complex as a stable component of Dicer/TRBP complex. We hypothesize that these three proteins form the core of a complex responsible for processing and execution of RNAi response. We also uncovered evidence indicating that the 60S ribosome subunit associates with TRBP to form a complex involved in mediating microRNA-directed translational repression. Aim 1 will define the function of Dicer/TRBP complex in the genesis of siRNA/miRNA and begin to examine the role of individual proteins and their domain structure in the regulation of siRNA/miRNA processing activity. Aim 2 describes the detailed functional analysis of the association of Dicer/TRBP complex with the effector complex containing the Ago2 subunit and delineates the role of individual proteins in the functioning of the posttranscriptional gene silencing. Aim 3 extends the work to the analysis of the large TRBP- containing complex, which is associated with the 60S ribosome subunit and the human Armitage protein involved in translational repression.