Characterization of TRBP-containing complexes

描述（由申请人提供）：秀丽隐杆线虫的microrna组基因需要控制幼虫发育和神经元模式的时间。这些基因中的大部分在哺乳动物中是保守的，最近的实验表明它们在调节造血分化和癌症中起作用。我们对microRNA通路的生化表征揭示了RNase III酶Dicer与致癌蛋白TRBP的关联。Dicer是负责生成成熟microRNA和将外源双链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与Dicer的关联，还发现了Argonaute 2作为Dicer/TRBP复合物的稳定组分存在，Argonaute 2是rna诱导沉默复合物的催化引擎。我们假设这三种蛋白质构成了负责处理和执行RNAi反应的复合体的核心。我们还发现了证据表明60S核糖体亚基与TRBP结合形成一个复合物，参与介导microrna导向的翻译抑制。目的1将定义Dicer/TRBP复合物在siRNA/miRNA发生中的功能，并开始研究单个蛋白质及其结构域结构在siRNA/miRNA加工活性调控中的作用。Aim 2描述了Dicer/TRBP复合体与含有Ago2亚基的效应复合体的详细功能分析，并描述了单个蛋白质在转录后基因沉默功能中的作用。Aim 3将工作扩展到分析含有TRBP的大复合体，该复合体与60S核糖体亚基和参与翻译抑制的人类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.