Internal Initiation in the Translation of Cellular mRNAs

细胞 mRNA 翻译的内部起始

• 批准号: 6636494
• 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/6636494
• 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（申请人摘要）：一些小核糖核酸病毒和细胞mRNA 在内部核糖体进入时以CAP独立方式启动翻译 在mRNA中包含的IRES位点。虽然在广泛的研究， 小核糖核酸病毒，知之甚少的内部启动在细胞的mRNA。 细胞内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， 细胞和微小瘤病毒IRES之间的联系毛罗实验室分析了两个 细胞IRES包含在编码Gtx的mRNA的5 'UTR内， 同源结构域蛋白和Rbm3，一种特殊的冷应激诱导蛋白， 因为它似乎增强了自己的IRES的活动。这些 研究表明，一些细胞IRE Ses由较短的 顺式作用调节序列，有些短至7个核苷酸， 独立运行以内部启动翻译（IRES模块），或 增强内部起始（增强子元件）。此外， IRES-模块从含有短随机核苷酸的文库中选择， 使用本实验室开发的方法进行测序。多个副本 一些自然发生的和选择的IRES结节增加了内部 协同启动。工作假设是一些细胞IRES 由较短的元件组成，可以独立工作。拟议 研究将确定天然存在的IRES模块和调控元件 而选择研究将试图识别序列， 具有不同的表达式属性。这些序列将被分析， 确定它们是否可以被分类，以调查管理它们的规则， 活动，并审查是否直接由3个招聘翻译机 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 一种反式作用蛋白。拟议的研究应提供新的见解 我们对内在启蒙的理解由于临床上有几个 重要的细胞基因包含IRES，这种增加的理解可能会使 有用的治疗操作。此外，使用IRE S模块和 增强子已经导致合成的IRES更短， 比目前使用的大病毒IRES更有效 在研究、基因治疗和 生物技术.