AUTOANTIGEN RNA HELICASE II/GU

最近，我们实验室用西瓜胃病患者的自身免疫血清克隆了一个编码核仁RNA解旋酶的基因。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‘到3’ATP依赖的RNA解离活性和GTP刺激的RNA折叠/退火活性。这两种活性是相反调节的，并位于同一多肽的不同结构域中。这是第一个被鉴定的哺乳动物核仁RNA解旋酶原型。电子显微镜显示其定位于核仁结构，与rRNA的转录、早期加工和成熟有关。假设RH-II/Gu参与rDNA的转录、结合和/或解离小核仁RNA，指导前rRNA的定点切割和修饰，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退火蛋白在前mRNA的加工过程中起到了很好的作用，但在哺乳动物前rRNA的加工过程中却没有。这一拟议项目的长期目标是确定RH-II/Gu的结构和功能特征。具体目的是(1)对RH-II/Gu蛋白的结构进行分析，包括其折叠结构域的晶体结构；(2)确定RH-II/Gu蛋白在核糖体RNA生物发生中的作用(S)；(3)研究RH-II/Gu识别的底物的结构特征。本研究的结果将为RH-II/Gu的结构特征和调控以及哺乳动物rRNA的产生机制提供有用的信息，rRNA是一种与前mRNA剪接相比特征较少的生化途径。Rh-II/Gu可能是一个很好的化疗靶点，该酶的结构特征可能会导致药物的开发。这项研究也可能为理解RH-II/Gu在诱导自身免疫反应中的病理作用提供帮助。

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.