Structure and function of the U3 RNA-protein complex

描述(由申请人提供)：真核核糖体生物发生正在成为癌症治疗的一个未被探索的目标。肿瘤抑制蛋白Rb、Art和P53同时下调细胞分裂和核糖体生物合成；反之，致癌蛋白c-Myc、Ras和PI3K上调这两个过程。核糖体生物发生的上调确保了达到临界细胞量不会限制细胞的增殖。重要的是，有效的化疗药物5-氟尿嘧啶抑制前体核糖体RNA(前rRNA)的加工，这是核糖体生物发生的关键步骤，因此也是细胞生长的关键步骤。长期目标是破译必需蛋白质及其组装如何指导核糖体生物发生的关键步骤中发生的动态RNA重排，即U3小核仁RNA(SnoRNA)的两个位点和前rRNA中的两个互补位点之间的杂交。这项工作将填补一个重大的知识空白，并是设计通过损害关键的生物发生步骤来抑制癌细胞增殖的治疗方法的先决条件。实现这一目标的一个障碍是缺乏适当的生化分析。为了克服这一障碍，PI开发了一种体外实验来监测U3-Pre-rRNA双链的形成，并确定ImpSp和Imp4p蛋白介导了这些双链的形成。中心假设是，ImpSp、Imp4p和U3 snoRNA之间的三元复合体降低了杂交障碍(例如双链不稳定性或隐藏的碱基配对位点)，以刺激U3-pre-rRNA双链的形成，并且这些蛋白质与其相互作用的伙伴MpplOp的组装调节单个蛋白质的活性。为了提供解决这些假设所需的热力学和动力学框架，PI开发了几种基于荧光共振能量转移的分析方法。Pi开发的分析方法使他的实验室处于一个独特的位置，可以促进对核糖体生物发生的了解，并通过追求四个目标来检验这些假说：(1)探索三元复合体降低(1)U3-Ets双链稳定的热力学屏障和(2)形成U3-18S双链的动力学屏障的分子机制；(3)描述三元复合体的组装和相互作用伙伴蛋白MpplOp的添加如何调节特定位点的退火活性；以及(4)通过X射线结晶学确定三元复合体的RNA结合和退火活性和组装的结构基础。

DESCRIPTION (provided by applicant): Eukaryotic ribosome biogenesis is emerging as an unexplored target for cancer treatment. The tumor- suppressor proteins Rb, Art and p53 simultaneously downregulate cell division and ribosome biogenesis; conversely the oncogenic proteins c-Myc, Ras and PI3K upregulate both processes. Upregulation of ribosome biogenesis ensures that reaching critical cell mass does not limit cell proliferation. Importantly, the effective chemotherapeutic agent 5-fluorouracil inhibits processing of the precursor ribosomal RNA (pre- rRNA), an essential step of ribosome biogenesis and hence of cellular growth. The long-term objective is to decipher how essential proteins and their assemblies direct the dynamic RNA rearrangements that occur during a key step of ribosome biogenesis; namely hybridization between two sites of the U3 small nucleolar RNA (snoRNA) and two complementary sites in the pre-rRNA. This work will fill a major knowledge gap and is a prerequisite for therapies designed to inhibit proliferation of cancer cells by impairing a key biogenesis step. A barrier to achieving this objective is a lack of appropriate biochemical assays. To surmount this barrier, the PI has developed an in vitro assay to monitor formation of the U3-pre-rRNA duplexes and determined that the proteins ImpSp and Imp4p mediate formation of these duplexes. The central hypotheses are that a ternary complex between ImpSp, Imp4p and the U3 snoRNA lowers hybridization barriers (e.g. duplex instability or a buried base pairing site) to stimulate formation of the U3-pre-rRNA duplexes and that assembly of these proteins with their interacting partner, MpplOp, modulates activities of the individual proteins. To provide the thermodynamic and kinetic framework needed to address these hypotheses the PI has developed several assays based on fluorescence resonance energy transfer. The assays developed by the PI place his laboratory in a unique position to advance understanding of ribosome biogenesis and test these hypotheses by pursuing the four aims: probe the molecular mechanism by which the ternary complex lowers the (1) thermodynamic barrier to stability of the U3-ETS duplex and (2) kinetic barrier to formation of the U3-18S duplex; (3) describe how assembly of the ternary complex and addition of the interacting partner protein MpplOp modulates the site-specific annealing activities; and (4) determine the structural basis of the RNA binding and annealing activity and assembly of the ternary complex by X-ray crystallography.