Characterization of TRBP-containing complexes

描述（由申请人提供）：需要秀丽隐杆线虫的 microRNA 基因组来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，最近的实验证明了它们在调节造血分化和癌症中的作用。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 以及将外源双链 RNA 加工成短干扰 RNA (siRNA) 的酶。对含有TRBP的复合物的生化分析不仅证实了TRBP和Dicer的关联，而且还揭示了Argonaute 2的存在，Argonaute 2是RNA诱导沉默复合物的催化引擎，是Dicer/TRBP复合物的稳定组分。我们假设这三种蛋白质构成了负责处理和执行 RNAi 反应的复合物的核心。我们还发现了证据表明 60S 核糖体亚基与 TRBP 结合形成复合物，参与介导 microRNA 指导的翻译抑制。目标 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在 siRNA/miRNA 加工活性调节中的作用。目标 2 描述了 Dicer/TRBP 复合物与含有 Ago2 亚基的效应复合物之间的关联的详细功能分析，并描述了各个蛋白质在转录后基因沉默功能中的作用。目标 3 将工作扩展到包含 TRBP 的大型复合物的分析，该复合物与 60S 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.