STRUCTURE AND FUNCTION OF YEAST SMALL NUCLEAR RNPS

小核糖核酸(SnRNAs)与前信使RNA相互作用 (前-信使核糖核酸)以建立剪接体的结构。一场激动人心的 剪接分析的复杂性正在显现：一些剪接体 在剪接过程中，SnRNA似乎经历了二级结构的变化。 为什么SnRNA重排对SnRNA功能很重要？ 拼接？这些重新安排是如何实现和监管的？什么 影响这些构象的启动和控制的因素 改变？ 我们将通过确定和描述 U2 SnRNP与剪接装置的其他组件的相互作用。 我们的假设是：与U2单链RNA相互作用的因子或介导 剪接体内的RNA结构相互转换反应 装配和功能在规则中起着至关重要的作用， 剪接反应的进展和准确性。我们将发挥作用 在剪接的调节、进展和准确性中起着至关重要的作用 反应。我们将1)描述与U2相互作用的组件， 剪接体组装和剪接过程中的SnRNA，2)探针变化 在SnRNA二级结构或因子与SnRNA的结合过程中 3)剪接过程中U2、Ut和Pre-mRNA的拓扑结构研究 拼接。 我们已经确定了与U2 SnRNA在遗传上相互作用的因素。 具有热敏感型prp 5、9、11或21等位基因的双突变株 某些U2等位基因(有些在野生型背景下没有表型)， 在通常允许生长的温度下死亡，或者在 变种人。我们已经分离出U3阴性的新突变 (太阳)，具有相关的温度敏感型剪接缺陷，并且 在任何已知的PRP基因中都没有。我们已经确定了感冒的抑制剂 定义新的新的敏感U2突变(CUS，冷敏感U2抑制因子) 调节U2功能的基因。我们将详细描述 这些基因产物与U2单链RNA的相互作用。 使用化学探针以对字幕敏感的方式修饰RNA RNA结构和化学环境的变化，我们将描述 不同的单链RNA采用的二级结构 继续进行拼接路径。这种方法将直接提供 这些变化的证据以及将这些变化与事件联系起来 在拼接方面。 最后，使用我们开发的一种简单的方法来生产大量 对于环状RNA，我们将研究U2， U6和剪接反应中的Pre-mRNA。 U2SnRNP与Pre-mRNA结合是剪接的关键早期步骤 途径，一条似乎有助于调节替代的途径 拼接。此外，U2SnRNA是必需的功能，将提供 剪接反应的第二催化步骤。关于酵母的知识 U2单链RNA功能将为理解 哺乳动物剪接器的功能和调节。

Small nuclear RNAs (snRNAs) interact with each other and premessenger RNA (pre-mRNA) to establish the structure of the spliceosomal. An exciting complexity in the analysis of splicing is emerging: some spliceosomal snRNAs appear to undergo secondary structure changes during splicing. Why and how are snRNA rearrangements important for snRNA function is splicing? How are these rearrangements achieved and regulated? What factors influence the initiation and control of these conformational changes? We will address these questions by identifying and characterizing the interaction of U2 snRNP with other components of the splicing apparatus. Our hypothesis is: factors that interact with U2 snRNA or mediated the RNA structural interconversion reactions within the spliceosome as it assembles and functions play an essential role in the regulation, progression and accuracy of splicing reactions. We will functions play an essential role in the regulation, progression and accuracy of splicing reactions. We will 1) characterize components that interact with U2, snRNA during spliceosome as it assembles and splicing, 2) probe changes in snRNA secondary structure or binding of factors to snRNAs during splicing, and 3)study of topology of U2, Ut, and the pre-mRNA during splicing. We have identified factors that interact genetically with U2 snRNA. Double mutant strains with heat sensitive prp 5, 9, 11 or 21 alleles and certain U2 alleles (some having no phenotype in a wild type background), die at temperatures normally permissive for growth or either single mutant. We have isolated new mutations that are suboptimal U3 negative (sun), have associated temperature sensitive splicing defects, and are not in any known prp gene. We have identified suppressor of a cold sensitive U2 mutation (CUS, cold sensitive U2 suppressor) that define new genes that modulate U2 function. We will characterize in detail the interaction of these gene products with U2 snRNA. Using chemical probes that modify RNA in a manner sensitive to subtitle changes in RNA structure and chemical environment, we will describe secondary structures adopted by different individual snRNAs as they proceed through the splicing pathway. This approach will provide direct evidence for such changes as well as relating these changes to the events in splicing. Finally, using a simple method we developed for producing large amounts of circular RNA, we will study the topological relationships between U2, U6, and pre-mRNA during the splicing reactions. U2 snRNP binding to pre-mRNA is a critical early step in the splicing pathway, one which appears to contribute to the regulation of alternative splicing. In addition, U2 snRNA is required function will provide a second catalytic steps of the splicing reaction. Knowledge about yeast U2 snRNA function will provide a sound conceptual basis for understanding the function and regulation of the splicing apparatus in mammals.