ASSEMBLY, LOCALIZATION AND FUNCTION OF THE U3 SNRNP

我们研究的长期目标是了解 在真核生物中前核糖体RNA（rRNA）加工中发生的事件， 特别是U3小型核武器的作用， 核糖核蛋白（snRNP）在初始的前rRNA切割事件。的U3 snRNP是一种丰富的snRNP，已被证明是必需的， 多种生物体中前rRNA加工中的第一次切割事件， 人类变成酵母在脊椎动物中，U3 snRNP由约200 核苷酸长度结合到几个蛋白质。虽然U3 snRNP 已经在多种物种和细胞类型中进行了研究，包括体内和体外研究。 在体外，其在前rRNA中作用的分子机制 加工过程在很大程度上仍然未知。具体而言，尚不清楚 蛋白质成分的组成对于其活性至关重要， 有助于U3 snRNP生物发生。 Fibrillarin是其他核仁snRNP共同的U3蛋白之一， 在全身性疾病患者中自身免疫反应的常见靶点 硬化症Fibrillarin是唯一的U3蛋白质，它以前是 详细研究其余五种或更多种蛋白质在 snRNP生物发生和pre-rRNA加工仍有待阐明。 为追求大会的性质，提出了五个具体目标， U3 snRNP的生物起源和功能： 1.纯化U3 snRNP及其单个蛋白组分， 哺乳动物来源 2.使用聚合酶链克隆编码U3蛋白的基因 反应 3.在大肠杆菌中表达U3蛋白，并使用融合蛋白衍生 特异性抗体 4.使用克隆和抗体： a.研究snRNA上U3蛋白的结构， B.研究U3 snRNP的细胞生物发生，包括蛋白质和 核仁定位所必需的RNA序列 5.研究pre-rRNA加工所必需的U3 snRNP组分 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.