Internal Initiation in the Translation of Cellular mRNAs

描述(申请人摘要)：一些微小核糖核酸病毒和细胞mRNAs 以独立于CAP的方式在内部iBosome条目处启动翻译 包含在信使核糖核酸中的位点(IRES)。在广泛研究的同时， 小核糖核酸病毒，对细胞内启动的mRNAs知之甚少。 细胞IRES的边界一直很难定义和排序 比较表明，细胞内的IRES或RRES之间没有明显的序列相似性 在细胞和微囊瘤病毒IREES之间。毛罗实验室分析了两个 包含在编码Gtx的mRNA5‘UTRs内的细胞IRES，a 同源结构域蛋白和冷应激诱导的特殊蛋白RBM3 这很有兴趣，因为它似乎增强了它自己的IRES的活动。这些 研究表明，一些细胞的IRSE由较短的 顺式作用的调控序列，有些甚至短到7个核苷酸，这可能 独立运行以在内部启动翻译(IRES模块)，或 增强内部启动(增强子元素)。此外，还有各种各样的 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一种方法进行测序。多个副本 一些自然发生的和精选的IRES结核在内部增加 协同启动。工作假说是一些细胞IRESE 由可以独立发挥作用的较短元素组成。建议数 研究将确定自然产生的IRES-模块和监管元素 来自细胞mRNA，而选择研究将试图识别序列 具有不同的表达式属性。这些序列将被分析以 确定它们是否可以分类，以调查管理其 活动，并检查3家公司是否直接招聘翻译机构 与rRNA或核糖体蛋白相互作用，或通过 中间蛋白。RBM3蛋白将作为一个潜在的例子进行研究 一种反式作用蛋白质。拟议的研究应提供新的见解 进入我们对内在启蒙的理解。因为有几个临床上 重要的细胞基因含有IRES，这一理解的增加可能会允许 有用的治疗手法。此外，IRE S模块的使用和 增强剂已经产生了更短、更多的合成IRES 比目前使用的IR双顺反子构建的大型病毒IRES更有效 应该在研究，基因治疗，以及 生物技术。

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.