Characterization of TRBP-containing complexes

描述(申请人提供)：线虫的microRNA基因组需要控制幼虫发育和神经元模式的时间。这些基因中的大多数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方面发挥了作用。我们对microRNA途径的生化特征表明，RNaseIII酶Dester与致癌蛋白TRBP有关。DICER是一种酶，负责产生成熟的microRNA，并将外源双链RNA加工成短干扰RNA(SiRNAs)。对含有TRBP的复合体进行生化分析，不仅证实了TRBP与DICER的结合，而且揭示了作为DICER/TRBP复合体的稳定组分的RNA诱导沉默复合体的催化引擎ArgAerte 2的存在。我们假设这三种蛋白质形成了负责处理和执行RNAi反应的复合体的核心。我们还发现了证据表明，60S核糖体亚单位与TRBP结合，形成一种复合体，参与介导microRNA导向的翻译抑制。目的1将确定DICER/TRBP复合体在siRNA/miRNA发生中的功能，并开始研究单个蛋白质及其结构域在调节siRNA/miRNA加工活性中的作用。目的2详细分析DICER/TRBP复合体与含有Ago2亚单位的效应复合体的结合，并阐明单个蛋白质在转录后基因沉默功能中的作用。目的3将工作扩展到含有TRBP的大型复合体的分析，该复合体与60S核糖体亚基和参与翻译抑制的人类Armitage蛋白有关。

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.