Structure/function of yeast small nuclear RNPs

描述（由申请人提供）：剪接在真核基因表达途径中的核心作用以及snrna在剪接体工作中的关键功能是众所周知的。但与RNA聚合酶和核糖体不同，剪接体只能通过低分辨率的视角来了解，其元素与功能的联系很少。许多悬而未决的问题仍然存在，如RNA解旋酶蛋白如何在剪接体的组装过程中促进RNA重排，剪接调节蛋白如何控制这些组装事件？为了揭开剪接体组装的奥秘，它的调控和它在系统水平上的整合，我们将采用实验上通用的芽殖酵母剪接机制作为一个模型，有三个特定的目的。在第一个目标中，我们将确定RNA解旋酶家族成员Prp5和RNA结合蛋白Cus2和Mud2如何促进pre-mRNA和U2在pre- plicosome组装过程中的重排。这三种蛋白质都有RNA结合域，但它们结合的确切RNA序列尚不清楚。在剪接复合体形成过程中，我们将捕获snRNA和结合它们的前mrna区域并对其进行测序。这一目标将回答关于pre-mRNA在组装过程中如何进入剪接体的重要问题。在第二个目标中，我们将学习Mer1如何通过内含子增强子促进剪接。我们将应用生化方法，包括体外剪接反应的重构，以及与Daniel Pomeranz Krummel （Brandeis）合作的结构方法，以及与Melissa J. Moore （HHMI/U）合作的单分子方法。Mass)和Jeff Gelles （Brandeis）。这一目标将定义剪接激活的分子事件，并为理解哺乳动物细胞中更复杂的剪接调控提供机制基础。在第三个目标中，我们将探索跨竞争控制的机制，作为在系统水平上调节剪接和mRNA代谢的新范式。我们在减数分裂过程中剪接的全局调控方面的工作揭示了剪接机制的前mrna竞争现象，并对细胞水平上的剪接调控机制具有重要意义。我们将检验U2 snRNP的稳定结合是竞争步骤的假设。这一目标将开始定义剪接调控中前体mrna竞争的参数和机制。酵母中遗传学、生物化学和基因组学的综合优势以及剪接机制的基本守恒表明，我们的努力将直接转化为对所有真核细胞中转录后基因调控机制的新理解。

DESCRIPTION (provided by applicant): The central role of splicing in the eukaryotic gene expression pathway and the critical function of snRNAs in the workings of the spliceosome are well known. But unlike RNA polymerase and the ribosome, the spliceosome is known only by low-resolution views whose elements are poorly connected to function. Many open questions remain such as how do RNA helicase proteins promote RNA rearrangements during assembly of the spliceosome, and how do splicing regulatory proteins control these assembly events? To unravel the mysteries of spliceosome assembly, its regulation and its integration at the systems level, we will employ the experimentally versatile budding yeast splicing machinery as a model with three specific aims. In the first aim we will determine how the RNA helicase family member Prp5 and the RNA-binding proteins Cus2 and Mud2 promote rearrangement of pre-mRNA and U2 during prespliceosome assembly. These three proteins have RNA binding domains, but which exact RNA sequences they bind are not known. We will capture and sequence snRNA and pre-mRNA regions that bind them as splicing complex formation proceeds. This aim will answer important questions about how the pre-mRNA is brought into the spliceosome during assembly. In the second aim we will learn how Mer1 promotes splicing through its intronic enhancer. We will apply biochemical methods including reconstitution of in vitro splicing reactions, as well as structural approaches in collaboration with Daniel Pomeranz Krummel (Brandeis) and a single-molecule method in collaboration with Melissa J. Moore (HHMI/U. Mass) and Jeff Gelles (Brandeis). This aim will define the molecular events of splicing activation and provide a mechanistic basis for understanding more complex splicing regulation in mammalian cells. In the third aim we will explore the mechanism of trans-competition control as a new paradigm for regulation of splicing and mRNA metabolism at the systems level. Our work on the global regulation of splicing during meiosis revealed the phenomenon of pre-mRNA competition for the splicing machinery, and bears critically on the mechanism of splicing regulation at a cellular level. We will test the hypothesis that stable binding of the U2 snRNP is the competitive step. This aim will begin to define the parameters and mechanisms of pre-mRNA competition in splicing regulation. The combined strengths of genetics, biochemistry, and genomics available in yeast and the fundamental conservation of the splicing machinery indicate that our efforts will translate directly to new understanding of the mechanisms of global posttranscriptional gene regulation in all eukaryotic cells.