Function of U1 snRNP in Formation of the Spliceosome

智力上的优点。大多数人类蛋白质编码基因包含主要的蛋白质编码区(外显子)，中间有非编码区(内含子)。在前体-mRNA剪接过程中，必须切除内含子，并将外显子剪接在一起，以产生适合翻译成蛋白质的mRNA转录本。前体-信使核糖核酸的剪接是由一个非常大且高度动态的“机器”--剪接体催化的。剪接体是通过组装到五个RNA-蛋白质复合体或U-SnRNP的前体-mRNA转录本上形成的。我们对U-SnRNPs如何启动剪接体组装和组织剪接体进行催化缺乏清楚的了解。这种认识上的差距在很大程度上是由于剪接体的巨大尺寸、动态特性以及使用粗细胞提取物进行研究的必要性所带来的实验挑战。本项目的目的是阐明U-SnRNPs组装到前体-mRNA转录本上的初始事件如何有助于形成表现出高保真的活性剪接体。为了实现这一长期目标，该项目将利用新的和创新的方法来“捕获”剪接体在其反应周期的早期中间状态，并帮助可视化其结构。这个项目的一个长期目标是为一项旨在激励学生从事科学并了解结构如何提供对功能的洞察的教育倡议奠定基础。利用新媒体，该项目旨在创建一个教育宣传活动，让高中生参与科学，并为他们的科学教师提供关于将新媒体和高分子结构融入课堂的培训和专业发展。

Intellectual merit. Most human protein-coding genes contain the primary protein-coding regions (exons) interrupted by non-coding regions (introns). In the process of precursor-mRNA splicing, the introns must be excised and the exons spliced together to generate an mRNA transcript suitable for translation into protein. Precursor-mRNA splicing is catalyzed by a remarkably large and highly dynamic "machine", the spliceosome. The spliceosome is formed by the assembly onto a precursor-mRNA transcript of five RNA-protein complexes or U snRNPs. We lack a clear understanding of how the U snRNPs initiate spliceosome assembly and organize the spliceosome for catalysis. This gap in knowledge is due in large part to experimental challenges posed by the spliceosome's large size, dynamic properties, and necessity to use crude cell extract for its study. The aim of this project is to elucidate how the initial events in the assembly of the U snRNPs onto a precursor-mRNA transcript contribute to formation of an active spliceosome that exhibits high fidelity. To accomplish this long-term goal, this project will utilize new and innovative methods designed to "trap" the spliceosome at early intermediate states of its reaction cycle and to aid the visualization of its structure.Broader impact. A long-term goal of this project is to lay the foundation for an educational initiative aimed at motivating students to engage in science and be exposed to how understanding structure provides insight into function. Using new media, this project aims to create an educational outreach that will engage high school students in the sciences and provide their science teachers with training and professional development on integration of new media and macromolecular structures into their classrooms.