Structure and activity of a cellular IRES element

描述（由申请人提供）：mRNA的翻译受到严格调控，需要起始因子和核糖体亚基的有序组装。某些病毒和细胞mrna利用称为内部核糖体进入位点（IRESs）的结构化RNA元件将核糖体引导到翻译起始位点，从而绕过了翻译起始的传统要求。IRES结构与功能密切相关，但对细胞IRES的三维组织却知之甚少。本研究旨在获得Cyr61 mRNA中IRES元件及其功能所需蛋白质复合物的晶体结构，提供细胞IRES元件的第一个高分辨率结构。Cyr61是一种血管生成诱导剂，被认为在肿瘤进展中起关键作用。双离子报告试验将定义最小的功能性IRES，并确定活性所需的交互作用因子。化学和酶探测将确定Cyr61 IRES的二级结构，并确定适合结晶的结构元素。这项工作的结果将产生对Cyr61 IRES的详细机制理解，并可能阐明细胞IRES活性的机制，可能确定治疗上操纵Cyr61产生的策略。

DESCRIPTION (provided by applicant): Translation of mRNA is tightly regulated, requiring the ordered assembly of initiation factors and ribosomal subunits. Certain viral and cellular mRNAs circumvent traditional requirements for translation initiation by utilizing structured RNA elements termed internal ribosome entry sites (IRESs) to direct ribosomes to the translational start site. IRES structure is intimately linked to function, but the three-dimensional organization of cellular IRESs is poorly understood. This proposal aims to obtain a crystallographic structure of the IRES element in Cyr61 mRNA in complex with proteins required for its function, providing the first high-resolution structure of a cellular IRES element. Cyr61 is an angiogenic inducer suggested to play a critical role in tumor progression. Dicistronic reporter assays will define a minimal functional IRES and trans-acting factors required for activity will be identified. Chemical and enzymatic probing will determine the secondary structure of the Cyr61 IRES and identify structural elements amenable to crystallization. Results from this work will yield a detailed mechanistic understanding of the Cyr61 IRES and may elucidate mechanisms of cellular IRES activity, possibly identifying strategies to therapeutically manipulate Cyr61 production.