THE MECHANISM AND FUNCTION OF A TO I RNA EDITING

A to I RNA 编辑的机制和功能

• 批准号: 6689587
• 负责人: JINGHUA YANG
• 金额: $ 20.91万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-01-01 至 2006-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6689587
• 关键词: HeLa cells; RNA binding protein; RNase protection assay; SDS polyacrylamide gel electrophoresis; active sites; complementary DNA; crosslink; gene expression; genetic regulation; immunoaffinity chromatography; messenger RNA; molecular cloning; neutralizing antibody; nucleic acid hybridization; nucleic acid structure; polymerase chain reaction; posttranscriptional RNA processing; protein isoforms; protein kinase; site directed mutagenesis; thin layer chromatography

Pre-mRNA editing is a recently discovered process that leads to the production of a variety of protein isoforms from a single gene. The A-to-I RNA editing, a distinct mechanism of mRNA editing in mammalian cells has been previously identified by our laboratory. Specifically, using HeLa cells we have shown that glutamate receptor subunit B (gluR-B) mRNA can undergo site specific deamination at an adenosine residue which converts adenosine to inosine (A-to-I), a critical change required to maintain receptor function. Two RNA-dependent adenosine deaminases (A-to-I RNA editase) have been identified in HeLa as well as a variety of other mammalian cells. In agreement with these observations, numerous inosine-containing mRNA (I-mRNA) were detected in different tissues as well. The presence of A- to-I RNA editase and I-mRNA in mammalian cells suggests that A- to-I RNA editing play an important role in gene expression and function. The broad, long-term objective of this proposal is to understand the mechanism and function of A-to-I RNA editing. Three specific aims are proposed as a basis for this research plan. First, to characterize I-mRNA in mammalian cells using the modified affinity purification approach to be established. Specifically, I-mRNA from HeLa cells will be enriched and identified. The cDNA of I-mRNA will be analyzed to determine the A-to-I editing site. The effect of the identified I-mRNA on protein identity, mRNA stability and structure will be studied in vitro and in vivo. Second, we will identify the site-specific interactions in the editase-RNA complex. Specifically, the conditions required for editase-RNA complex formation will be determined and intra-complex interactions between the editase and substrate and the catalytic core will be studied by site-specific UV cross-linking and base modifications. The possibility that the catalytic and RNA binding domains are functionally independent will be examined. Also, A-to-I RNA editase with a different RNA binding domain will be characterized, and protein factors associated with A-to-I RNA editase will be studied. Third, we will study the functions of A-to-I RNA editase in HeLa cells. Specifically, dominate-negative mutant and neutralizing antibody will be produced to establish a HeLa cell line in which editase activity can be quantitatively controlled. By using this cell line, the hypothesis that the dsRNA-dependent protein kinase (PKR) is mediated by the dsRNA editing activity of A-to-I RNA editase will be examined. The overall cellular effects of gene expression by A-to-I RNA editase and the I-mRNA decay will be studied. The proposed experiments will provide fundamental information regarding the role of A-to-I pre-mRNA editing in mammalian gene expression and regulation that could be used to combat disease.

前 mRNA 编辑是最近发现的一个过程，它可以从单个基因产生多种蛋白质亚型。 A-to-I RNA 编辑是哺乳动物细胞中 mRNA 编辑的一种独特机制，我们的实验室此前已发现该机制。 具体来说，我们使用 HeLa 细胞证明，谷氨酸受体亚基 B (gluR-B) mRNA 可以在腺苷残基处进行位点特异性脱氨，从而将腺苷转化为肌苷（A 至 I），这是维持受体功能所需的关键变化。 在 HeLa 以及多种其他哺乳动物细胞中已鉴定出两种 RNA 依赖性腺苷脱氨酶（A-to-I RNA 编辑酶）。 与这些观察结果一致，在不同的组织中也检测到了大量含有肌苷的 mRNA (I-mRNA)。 哺乳动物细胞中 A-to-I RNA 编辑酶和 I-mRNA 的存在表明 A-to-I RNA 编辑在基因表达和功能中发挥着重要作用。 该提案的广泛、长期目标是了解 A-to-I RNA 编辑的机制和功能。提出三个具体目标作为本研究计划的基础。 首先，使用待建立的改进的亲和纯化方法来表征哺乳动物细胞中的I-mRNA。具体来说，来自 HeLa 细胞的 I-mRNA 将被富集和鉴定。 将分析 I-mRNA 的 cDNA 以确定 A 到 I 的编辑位点。 将在体外和体内研究已鉴定的 I-mRNA 对蛋白质特性、mRNA 稳定性和结构的影响。 其次，我们将确定编辑酶-RNA 复合物中的位点特异性相互作用。 具体来说，将确定编辑酶-RNA复合物形成所需的条件，并通过位点特异性UV交联和碱基修饰来研究编辑酶和底物以及催化核心之间的复合物内相互作用。 将检查催化结构域和 RNA 结合结构域在功能上独立的可能性。 此外，还将对具有不同 RNA 结合域的 A-to-I RNA 编辑酶进行表征，并研究与 A-to-I RNA 编辑酶相关的蛋白质因子。第三，我们将研究A-to-I RNA编辑酶在HeLa细胞中的功能。 具体来说，将产生显性失活突变体和中和抗体，以建立可以定量控制编辑酶活性的HeLa细胞系。 通过使用该细胞系，将检验 dsRNA 依赖性蛋白激酶 (PKR) 由 A-to-I RNA 编辑酶的 dsRNA 编辑活性介导的假设。 将研究 A-to-I RNA 编辑酶和 I-mRNA 衰减对基因表达的总体细胞影响。拟议的实验将提供有关 A-to-I mRNA 前体编辑在哺乳动物基因表达和调节中的作用的基本信息，可用于对抗疾病。