Cell Proteins Required for Function of the IRES

描述(申请人提供)：建议进行细胞鉴定实验 猪瘟病毒内部核糖体进入位点(IRES)功能所需的蛋白质 丙型肝炎病毒(丙型肝炎)、脊髓灰质炎病毒(PV)、脑心肌炎病毒(EMCV)、 和酿酒酵母TFIID基因。IRES是高度有序的RNA序列 病毒和细胞中允许核糖体启动的mRNAs的5‘端 翻译不依赖于IIIRNA的5‘端。IRES功能取决于 典型的翻译起始因子以及其他细胞蛋白。 酵母酿酒酵母将被用来鉴定细胞蛋白 是IRESS功能所必需的。目标1：IREES的功能表达 酿酒酵母。编码双顺反子mRNA的DNA将被构建为 由ADE3基因、IRES和LacZ基因组成。Ade3p 蛋白质将通过5‘端依赖的翻译产生，并作为一种 存在该信使核糖核酸的标记。B-半乳糖苷酶将由 内部核糖体结合，并将作为IRES功能的标志。 含有该质粒的酿酒酵母将被诱变，而 产生降低水平的B-半乳糖苷酶将被确定。这些酵母 菌株可能包含对人类免疫功能至关重要的基因突变。 IRES的功能。 目的2：鉴定IRES功能所需的酿酒酵母基因。突变型 在AIM 1中鉴定的酵母菌株可能有一种基因突变，该基因是 对IRES功能很重要。为了鉴定突变基因，我们将引入一种 以质粒为基础的酿酒酵母基因组DNA文库导入突变株 弥补IRES在功能上的缺陷。核苷酸序列分析 将用于从酿酒酵母基因组中鉴定互补基因 数据库。 目的3：确定细胞蛋白在IRES功能中的作用。酵母菌 在AIM 2中发现的基因将被干扰，以正式证明编码的 IRES功能需要蛋白质。如果酵母蛋白结合到 不同的IRES，IRES上的结合位点将由 诱变。酿酒酵母基因在哺乳动物中的同源序列 将确定病毒IRES的功能，以及它们在IRES中的作用 功能将通过耗尽无细胞中的蛋白质来评估 翻译提取物和活细胞。这些研究将有助于 我们对IRESes功能的理解，IRESes是细胞和 病毒式增长。

DESCRIPTION (provided by applicant): Experiments are proposed to identify cell proteins required for function of the internal ribosome entry site (IRES) of hepatitis C virus (HCV), poliovirus (PV), encephalomyocarditis virus (EMCV), and the S. cerevisiae TFIID gene. IRESes are highly ordered RNA sequences at the 5' end of viral and cellular mRNAs that permit ribosomes to initiate translation independent of the 5'-end of the IIIRNA. IRES function depends on the canonical translation initiation factors as well as other cell proteins. The yeast Saccharomyces cerevisiae will be used to identify cell proteins that are required for function of IRESes. Aim 1: Functional expression of IRESes in S. cerevisiae. DNA encoding a bicistronic mRNA will be constructed that consists of the ADE3 gene followed by the IRES and the lacZ gene. The Ade3p protein will be produced by 5'-end dependent translation, and serves as a marker for the presence of the mRNA. B-galactosidase will be produced by internal ribosome binding, and will serve as a marker for function of the IRES. S. cerevisiae containing this plasmid will be mutagenized, and colonies that produce reduced levels of B-galactosidase will be identified. These yeast strains potentially contain mutations in genes that are important for the function of the IRES. Aim 2: Identification of S. cerevisiae genes required for IRES function. Mutant yeast strains identified in aim 1 presumably have a mutation in a gene that is important for IRES function. To identify the mutant genes, we will introduce a plasmid-based genomic DNA library of S. cerevisiae into the mutant strains to complement the defect in function of the IRES. Nucleotide sequence analysis will be used to identify the complementing gene from the S. cerevisiae genome database. Aim 3: Determination of the roles of cell proteins in IRES function. Yeast genes identified in aim 2 will be disrupted to formally prove that the encoded proteins are required for IRES function. If the yeast proteins bind to the different IRESes, the binding site on the IRES will be identified by mutagenesis. Mammalian sequence homologs of the S. cerevisiae genes important for function of the viral IRESes will be identified, and their role in IRES function will be assessed by depletion of the protein from cell-free translation extracts and from living cells. These studies will contribute to our understanding of the function of IRESes, important regulators of cell and viral growth.