Investigating the role of nuclear speckles in mRNP maturation

项目摘要/摘要 哺乳动物的细胞核由许多无膜细胞器(MLO)细分，这些细胞器与 围绕核质发挥不同的功能。核斑点是最突出的MLO之一 哺乳动物细胞核，每个细胞有20-50个斑点，每个斑点直径约1微米。斑点中有数百颗 参与几个mRNP成熟事件的RNA结合蛋白(RBPs)-包括前mRNA剪接和 MRNP核输出--而且在细胞核中也含有最高浓度的转录后RNA。 斑点的许多成分都是癌基因，而调节斑点的药物是抗肿瘤的。还没有 尽管它们在50多年前就被发现了，但斑点的分子功能尚不清楚。体内的核糖核酸 斑点还没有定义，也不清楚斑点是如何影响RNA加工或mRNP组装的。这 Proposal使用新的策略来阐明斑点的组成、调节和功能，并将 直接测试斑点影响mRNP成熟和改变哺乳动物基因表达的假设。 这项工作的前提是研究散斑的功能需要研究整个散斑， 而不仅仅是研究它们内部的个别因素。目标1将导致形成一个健壮的斑点- 一种使用荧光颗粒分选从细胞裂解物中分离GFP标记斑点的纯化方法。 对纯化的斑点进行定量蛋白质组学和RNA测序将提供第一个全面的分析 它们的组成。随后的分析将确定哺乳动物mRNPs流量的全部或子集 通过斑点，并将定义RNA是否由于特定的序列和/或 拼接不完整。目标2将评估斑点如何直接影响mRNP的成熟。自然而然的两种激酶 在不同的生理环境中促进斑点分解将分别被过度激活或抑制， 然后是全基因组分析的前mRNA剪接和核输出。确定以下共同影响 两个激酶将揭示斑点分解如何影响mRNP成熟，而不是每一个的其他影响。 个体激活剂。目标3将定义基本限制性商业惯例斑点内的RNA-蛋白质相互作用。UV- 在斑点纯化之后的交联将识别两个SR蛋白质的斑点特异性RNA相互作用， 一个限制性商业惯例家族，促进数以千计的mRNA的剪接和输出。这一分析将揭示 斑点内的限制性商业惯例与未剪接、交替剪接或连续剪接的底物RNA结合 异构体。最终，这项工作产生的方法和结果将作为调查的平台 斑点在发育和疾病的背景下。拟议的研究目标将提供重要的培训 使用许多全基因组方法，包括rna-seq、蛋白质组学和eCLIP，以及培训 人类细胞的现代基因操作。这项研究将在Karla Neugebauer博士的实验室进行， 耶鲁大学杰出的RNA生物学家，提供专业和科学指导。

Project Summary/Abstract Mammalian nuclei are subdivided by a host of membrane-less organelles (MLOs) that separate from the surrounding nucleoplasm to serve distinct functions. Nuclear speckles are among the most prominent MLOs in mammalian nuclei, with 20-50 speckles per cell, each measuring ~1 µm in diameter. Speckles contain hundreds of RNA binding proteins (RBPs) involved in several mRNP maturation events – including pre-mRNA splicing and mRNP nuclear export – and also contain the highest concentration of post-transcriptional RNA in the nucleus. Many components of speckles are oncogenes, and drugs that modulate speckles are anti-tumorigenic. Yet despite their discovery over 50 years ago, the molecular function of speckles is unknown. The RNA within speckles has not been defined, nor is it clear how speckles affect RNA processing or mRNP assembly. This proposal employs novel strategies to elucidate the composition, regulation, and function of speckles, and will directly test the hypothesis that speckles impact mRNP maturation and alter mammalian gene expression. The premise of this work is that investigating the function of speckles requires the study of entire speckles, and not just the study of individual factors within them. Aim 1 will result in the development of a robust speckle- purification method that uses fluorescent-particle sorting to isolate GFP-labeled speckles from cell lysate. Quantitative proteomics and RNA-sequencing of purified speckles will provide the first comprehensive analysis of their composition. Subsequent analysis will determine whether all or a subset of mammalian mRNPs traffic through speckles, and will define whether RNAs are detained in speckles due to specific sequences and/or incomplete splicing. Aim 2 will assess how speckles directly impact mRNP maturation. Two kinases that naturally promote speckle disassembly in distinct physiological contexts will be separately hyperactivated or inhibited, followed by genome-wide analysis of pre-mRNA splicing and nuclear export. Identifying common effects between two kinases will reveal how speckle disassembly impacts mRNP maturation, as opposed to other effects of each individual kinase. Aim 3 will define the RNA-protein interactions within speckles for essential RBPs. UV- crosslinking followed by speckle-purification will identify speckle-specific RNA interactions for two SR proteins, a family of RBPs that promote splicing and export of thousands of mRNAs. This analysis will reveal whether RBPs within speckles bind to substrate RNAs that are unspliced, alternatively spliced, or consitutively spliced isoforms. Ultimately, the methods and results stemming from this work will serve as a platform to investigate speckles in the context of development and disease. The proposed research goals will provide significant training in the use of many genome-wide approaches, including RNA-seq, proteomics, and eCLIP, as well as training in modern genetic manipulations of human cells. The research will take place in the lab of Dr. Karla Neugebauer, an outstanding RNA biologist at Yale University who provides both professional and scientific mentorship.