AUTOANTIGEN RNA HELICASE II/GU

自身抗原 RNA 解旋酶 II/GU

• 批准号: 6524208
• 负责人: BENIGNO C VALDEZ
• 金额: $ 19.01万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-01 至 2004-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6524208
• 关键词: RNA biosynthesis; RNase protection assay; Xenopus oocyte; autoantigens; binding sites; chemical kinetics; double stranded RNA; enzyme activity; enzyme mechanism; enzyme structure; enzyme substrate; expression cloning; genetic transcription; guanosine triphosphate; helicase; northern blottings; nucleic acid structure; nucleolus; posttranscriptional RNA processing; ribosomal RNA; site directed mutagenesis

A cDNA that codes for a nucleolar RNA helicase has recently been cloned in our laboratory using an autoimmune serum from a patient with watermelon stomach disease. Later studies in our laboratory show that this autoantibody is also present in some patients with connective tissue diseases. This 100-kDa autoantigen, referred to as RH-II/Gu protein, is a unique bifunctional enzyme which possesses a 5' to 3' ATP-dependent RNA unwinding activity and a GTP-stimulated RNA folding/annealing activity. The two activities are oppositely regulated and reside in separate domains of the same polypeptide. This is the first mammalian prototype nucleolar RNA helicase to be identified. Electron microscopy shows its localization to nucleolar structures associated with transcription, early processing and maturation of rRNA. It is hypothesized that RH-II/Gu is involved in rDNA transcription, binding and/or unwinding of small nucleolar RNAs which direct site-specific cleavages and modifications of the pre-rRNA, or in the proper folding of rRNA prior to binding of ribosomal proteins. The roles of RNA helicases and RNA annealing proteins are well-established in the processing of pre-mRNA, but not in the processing of mammalian pre-rRNA. The long term objective of this proposed project is to do structural and functional characterizations of RH-II/Gu. The specific aims are (1) to do structural analyses of the RH-II/Gu protein including crystal structure of its RNA folding domain, (2) to determine the role(s) of RH-II/Gu protein in ribosomal RNA biogenesis, and (3) to examine structural features of the substrates recognized by RH-II/Gu. The results from this study will provide useful information on structural characterization and regulation of RH-II/Gu, and on the mechanism of mammalian rRNA production, a less characterized biochemical pathway compared to pre-mRNA splicing. RH-II/Gu may be an excellent chemotherapeutic target, and structural characterization of this enzyme may lead to drug development. This study may also provide an impetus for understanding the pathological roles of RH-II/Gu on the elicitation of autoimmune responses.

最近，我们的实验室利用西瓜胃病患者的自身免疫血清克隆了一个编码核仁RNA解旋酶的cDNA。我们实验室后来的研究表明，这种自身抗体也存在于一些结缔组织疾病患者中。这种100 kda的自身抗原，被称为RH-II/Gu蛋白，是一种独特的双功能酶，具有5‘至3’ atp依赖的RNA解绕活性和gtp刺激的RNA折叠/退火活性。这两种活性受到相反的调控，位于同一多肽的不同结构域。这是第一个被鉴定的哺乳动物核仁RNA解旋酶原型。电镜显示其定位于与rna转录、早期加工和成熟相关的核仁结构。据推测，RH-II/Gu参与rDNA转录、小核仁rna的结合和/或解绕，从而指导pre-rRNA的位点特异性切割和修饰，或者参与rRNA在与核糖体蛋白结合之前的适当折叠。RNA解旋酶和RNA退火蛋白的作用在前mrna的加工中得到了证实，但在哺乳动物前rrna的加工中还没有得到证实。本项目的长期目标是对RH-II/Gu进行结构和功能表征。具体目的是：(1)对RH-II/Gu蛋白进行结构分析，包括其RNA折叠域的晶体结构；(2)确定RH-II/Gu蛋白在核糖体RNA生物发生中的作用；(3)检查RH-II/Gu识别的底物的结构特征。这项研究的结果将为RH-II/Gu的结构表征和调控提供有用的信息，以及哺乳动物rRNA产生的机制，这是一种与pre-mRNA剪接相比较少被表征的生化途径。RH-II/Gu可能是一个很好的化疗靶点，这种酶的结构表征可能会导致药物的开发。这项研究也可能为理解RH-II/Gu在自身免疫反应引发中的病理作用提供动力。