Mechanism of IRES-Mediated Translation Initiation

描述（由申请人提供）：大多数信使rna （mrna）是通过帽依赖的起始机制翻译的。然而，约5%至10%的mrna使用另一种启动蛋白质合成的机制，利用内部核糖体进入位点（IRES）将核糖体招募到信息中。IRESs存在于编码参与细胞生长、增殖、凋亡、缺氧和血管生成的蛋白质的mrna中。由于许多参与肿瘤发生的细胞mrna都含有ires，我们预计ires介导的翻译是开发抗癌疗法的一个很好的靶点，它将广泛适用于几种类型的癌症。由于其机制尚不清楚，我们对IRESs如何招募核糖体很感兴趣。多项研究表明，蟋蟀麻痹病毒（CrPV）基因间区IRES （IGR-IRES）与细胞IRES结合核糖体有一定的共同机制，提示IGR-IRES是一个很好的IRES模型。由于酵母和哺乳动物细胞之间的翻译高度保守，我们在酵母中开发了一个遗传系统来回答以下问题：1)哪些IGR-IRES结构或序列对IRES功能重要？2) IGR-IRES介导的翻译需要核糖体的哪些成分？3)这些接触如何促进核糖体结合？4) rRNA修饰对IRES活性的贡献是什么？5)这些发现如何应用于哺乳动物细胞的细胞IRESs ？这个系统为我们提供了遗传学、生物化学，并允许我们以在哺乳动物细胞中不可能的方式操纵翻译机制的组成部分。我们选择使用一种创新的遗传方法来理解IRESs介导的翻译，因为尽管多个实验室多年来的努力，细胞IRESs启动的机制仍然难以捉摸。

DESCRIPTION (provided by applicant): The majority of messenger RNAs (mRNAs) are translated by a cap-dependent mechanism of initiation. However, about 5% to 10% of mRNAs use an alternative mechanism of initiating protein synthesis, utilizing an internal ribosome entry site (IRES) to recruit the ribosomes to the message. IRESs are found in mRNAs that encode proteins involved in cell growth, proliferation, apoptosis, hypoxia, and angiogenesis. Because many cellular mRNAs that are involved in tumorigenesis contain IRESs, we anticipate that IRES-mediated translation is a good target for the development of anti-cancer therapies that will be broadly applicable to several types of cancer. Since the mechanism is not understood we are interested in how IRESs recruit ribosomes. Several studies have suggested that the cricket paralysis virus (CrPV) intergenic region IRES (IGR-IRES) and cellular IRESs share some mechanisms for binding ribosomes suggesting that the IGR-IRES is a good model IRES. Because translation is highly conserved between yeast and mammalian cells we have developed a genetic system in yeast to answer the following questions: 1) Which IGR-IRES structures or sequences are important for IRES function? 2) What components of the ribosome are required for IGR-IRES mediated translation? 3) How do these contacts facilitate ribosome binding? 4) What is the contribution of rRNA modifications for IRES activity? 5) How do these findings apply to cellular IRESs in mammalian cells? This system provides us with genetics, biochemistry, and will allow us to manipulate components of the translational machinery in ways that are not possible in mammalian cells. We chose to use an innovative genetic approach to understand IRES-mediated translation because the mechanism of initiation by cellular IRESs has been elusive despite many years of effort by multiple laboratories. PUBLIC HEALTH RELEVANCE: Some viral and cellular mRNAs utilize an alternative mechanism of translation initiation that recruits ribosomes internally to the message using a highly structured internal ribosome entry site (IRES). IRES-mediated translation of cellular mRNAs is involved in regulating cancer, cell death, cell growth, and angiogenesis, as well as translation of viral RNAs. We are using yeast genetics to understand the mechanism of IRES-mediated translation initiation.