Internal Initiation in the Translation of Cellular mRNAs

细胞 mRNA 翻译的内部起始

• 批准号: 6520303
• 负责人: VINCENT P MAURO
• 金额: $ 29.63万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-05-01 至 2005-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6520303
• 关键词: cell free system; gel mobility shift assay; genetic library; genetic regulatory element; genetic translation; intracellular transport; messenger RNA; molecular cloning; nucleic acid sequence; ribosomes; stress proteins

DESCRIPTION (applicant's abstract): Some picornaviral and cellular mRNAs initiate translation in a CAP-independent manner at internal ibosome entry sites (IRESes) contained within the mRNA. While extensively studied in picornaviruses, little is known about internal initiation in cellular mRNAs. The boundaries of cellular IRESes have been difficult to define and sequence comparisons show no obvious sequence similarities among cellular IRESes or between cellular and picomaviral IRESes. The Mauro laboratory has analyzed two cellular IRESes contained within the 5' UTRs of the mRNAs that encode Gtx, a homeodomain protein and Rbm3, a cold stress induced protein that is of special interest because it appears to enhance the activity of its own IRES. These studies indicated that some cellular IRE Ses were composed of shorter cis-acting regulatory sequences, some as short as 7-nucleotides, that could function independently to internally initiate translation (IRES-module), or to enhance internal initiation (enhancer element). In addition, a diversity of IRES-modules was selected from a library containing short random nucleotide sequences using a method that was developed in this laboratory. Multiple copies of some naturally-occurring and selected IRESnodules increased internal initiation synergistically. The working hypothesis is that some cellular IRESes are composed of shorter elements that can function independently. The proposed studies will identify naturally-occurring IRES-modules and regulatory elements from cellular mRNAs, while selection studies will attempt to identify sequences with different expression properties. These sequences will be analyzed to determine if they can be categorized, to investigate the rules governing their activity, and to examine if the3 recruit the translation machinery directly by interacting with rRNA or with ribosomal proteins, or indirectly through intermediary proteins. The Rbm3 protein will be studied as a potential example of a trans-acting protein. The proposed studies should provide new insights into our understanding of internal initiation. Inasmuch as several clinically important cellular genes contain IRESes, this increased understanding may allow useful therapeutic manipulations. Moreover, the use of IRE S-modules and enhancers has already resulted in synthetic IRESes that are shorter and more efficient than the large viral IRESes currently used ir dicistronic constructs and should have broad applications in research, gene therapy, and biotechnology.

描述（申请人的摘要）：一些小核糖核酸病毒和细胞 mRNA 在内部核糖体入口处以独立于 CAP 的方式启动翻译 mRNA 中包含的位点 (IRES)。在广泛研究的同时 对于小核糖核酸病毒，人们对细胞 mRNA 的内部起始知之甚少。 细胞 IRES 的边界很难定义和排序 比较显示细胞 IRES 之间没有明显的序列相似性或 细胞和小RNA病毒IRES之间。 Mauro 实验室分析了两种 细胞 IRES 包含在编码 Gtx 的 mRNA 的 5' UTR 内， 同源结构域蛋白和 Rbm3，一种具有特殊功能的冷应激诱导蛋白 兴趣，因为它似乎增强了其自身 IRES 的活动。这些 研究表明，一些细胞 IRE Ses 由较短的 顺式作用调控序列，有些短至 7 个核苷酸， 独立功能以内部启动翻译（IRES 模块），或 增强内部启动（增强子元件）。此外，多样化的 IRES 模块选自包含短随机核苷酸的文库 使用本实验室开发的方法进行序列。多份 一些天然存在的和选定的 IRES 结节增加了内部 协同启动。工作假设是一些细胞 IRES 由可以独立运行的较短元素组成。拟议的 研究将确定自然存在的 IRES 模块和监管要素 来自细胞 mRNA，而选择研究将尝试识别序列 具有不同的表达属性。这些序列将被分析 确定它们是否可以被分类，调查管理它们的规则 活动，并检查 the3 是否直接招募翻译机器 与 rRNA 或核糖体蛋白相互作用，或间接通过 中间蛋白。 Rbm3 蛋白将作为一个潜在的例子进行研究 的反式作用蛋白。拟议的研究应该提供新的见解 进入我们对内部启动的理解。由于临床上有几个 重要的细胞基因含有IRES，这种增加的了解可能允许 有用的治疗操作。此外，使用 IRE S 模块和 增强子已经产生了更短、更多的合成 IRES 比目前使用的大型病毒IRESes双顺反子构建体更有效 并应在研究、基因治疗和 生物技术。