ASSEMBLY, LOCALIZATION AND FUNCTION OF THE U3 SNRNP

我们研究的长期目标是了解分子 在真核生物的核糖体前RNA(RRNA)加工中发生的事件， 特别关注U3小核的作用 核糖核蛋白(SnRNP)在最初的Pre-rRNA切割事件中。U3 SnRNP是一种丰富的SnRNP，已被证明是 多个生物体中前rRNA加工中的第一个切割事件，来自 从人类到酵母。在脊椎动物中，U3SnRNP由约200个RNA组成 长度上的核苷酸与几种蛋白质结合。尽管U3的SnRNP已经 已经在各种各样的物种和细胞类型中进行了研究，包括体内和 在体外，其作用于Pre-rRNA的分子机制 处理过程在很大程度上仍是未知的。特别是，还不清楚是哪一种 对它的活性或它们如何 对U3SnRNP的生物发生做出贡献。 Fibrillarin是其他核仁SnRNPs共有的U3蛋白之一，它是 系统性红斑狼疮患者自身免疫反应的常见靶点 硬化症。纤维蛋白是唯一的U3蛋白 进行了详细的研究。剩下的五种或更多蛋白质在其中的作用(S) SnRNP的生物发生和前rRNA的加工仍有待阐明。 提出了五个具体目标，以追求大会的性质， U3SnRNP的生物发生和功能： 1.分离纯化U3单链RNP及其单项蛋白质组分 来自哺乳动物的。 2.利用聚合酶链法克隆编码U3蛋白的基因 反应。 3.在大肠杆菌中表达U3蛋白，并利用融合蛋白获得 特定的抗体。 4.使用克隆和抗体： A.研究SnRNA上U3蛋白的组织结构， B.研究U3SnRNP的细胞生物发生，包括蛋白质和 核仁定位所需的RNA序列 5.研究Pre-rRNA加工所必需的U3 SnRNP成分 使用体外处理系统。

The long term objective of our research is to understand the molecular events that occur in pre-ribosomal RNA (rRNA) processing in eukaryotes, focusing particularly of the role of the U3 small nuclear ribonucleoprotein (snRNP) in the initial pre-rRNA cleavage event. The U3 snRNP, one of the abundant snRNPs, has been shown to be essential for the first cleavage event in pre-rRNA processing in multiple organisms, from humans to yeast. In vertebrates, the U3 snRNP consists of an RNA about 200 nucleotides in length bound to several proteins. Although the U3 snRNP has been studied in a wide variety of species and cell types, both in vivo and in vitro, the molecular mechanism underlying its role in pre-rRNA processing remains largely unknown. In particular, it is not clear which of the protein components are critical for its activity or how they contribute to U3 snRNP biogenesis. Fibrillarin, one of the U3 proteins common to other nucleolar snRNPs, is a frequent target of the autoimmune response in patients with systemic sclerosis. Fibrillarin is the only U3 protein that has been previously studied in detail. The role(s) of the remaining five or more proteins in snRNP biogenesis and pre-rRNA processing remain to be elucidated. Five specific aims are proposed to pursue the nature of the assembly, biogenesis and function of the U3 snRNP: 1. Purify the U3 snRNP and its individual protein components from a mammalian source. 2. Clone the genes encoding the U3 proteins using the polymerase chain reaction. 3. Express the U3 proteins in E.coli and use the fusion proteins to derive specific antibodies. 4. Use the clones and antibodies to: a. Study the organization of the U3 proteins on the snRNA, b.Study the cellular biogenesis of the U3 snRNP, including proteins and RNA sequences necessary for nucleolar localization 5. Investigate the U3 snRNP components essential for pre-rRNA processing using an in vitro processing system.