Functional Coupling of Steps in Gene Expression

基因表达步骤的功能耦合

• 批准号: 7862779
• 负责人: ROBIN E. REED
• 金额: $ 58.61万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-15 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7862779
• 关键词: 3' Splice Site; ATP phosphohydrolase; Affect; Binding; Biochemical; Biological Assay; Biological Models; Boxing; Cell Nucleus; Cell physiology; Cis-Acting Sequence; Code; Complex; Coupled; Coupling; Cytoplasm; Defect; Diagnosis; Disease; Elements; Elongation Factor; Ensure; Exons; Gene Expression; Genes; Genetic Transcription; Goals; Introns; Laboratories; Length; Location; Mammalian Cell; Mediating; Messenger RNA; Modeling; Oncogene Proteins; Pathway interactions; Polyadenylation; Process; Proteins; Quality Control; RNA Interference; RNA Polymerase II; RNA Processing; RNA Splicing; Recruitment Activity; Research; Role; Site; Spliceosome Assembly Pathway; System; Testing; Transcription Elongation; Translations; U1 Small Nuclear Ribonucleoprotein; U2 Small Nuclear Ribonucleoprotein; helicase; human disease; in vivo; mRNA Export; mRNA Precursor; protein expression; protein function

Project Summary Expression of protein-coding genes is a multi-step process beginning with RNA polymerase II transcription and RNA processing in the nucleus followed by export of the mature mRNA to the cytoplasm for translation. All of the steps in gene expression are coordinated via an extensive network of both physical and functional interactions between the machineries that carry out each step of the pathway. Defects in gene expression are a major cause of human disease, and conversely, gene expression is one of the major cellular processes that can be harnessed to diagnose and treat disease. Thus, a detailed understanding of gene expression is essential for both understanding and treating disease. The long-term objective of the proposed research is to achieve a detailed understanding of the mechanisms for coupling transcription to splicing and for coupling splicing to mRNA export. In addition, the mechanisms and factors required for export of mRNAs derived from genes that naturally lack introns is a central objective. In Specific Aim 1, a coupled RNAP II transcription/splicing system will be established to use in conjunction with RNA interference to identify proteins that function in coupling. The oncoprotein TLS/FUS is a strong candidate protein that will be tested. The system will also be used to test a new model, the U1/SR stamping model, which proposes that the co-transcriptional recruitment of U1 snRNP and SR proteins to the 5¿ splice site functions to achieve the extremely high fidelity required for splicing. An immobilized coupled transcription/splicing assay will also be established in order to investigate the mechanisms that function in co-transcriptional RNA processing. Finally, the recent exciting discovery that transcription elongation factors are specifically associated with U2 snRNP will be investigated to determine whether addition of U2 snRNP to the pre-mRNA during spliceosome assembly is coupled to transcription elongation. In Specific Aim 2, the mechanism for coupling mRNA export to splicing will be investigated by combining biochemical studies with a powerful system for assaying mRNA export in mammalian cells. The function of the highly conserved mRNA export machinery (the TREX complex) will be determined using this system. The role of ATP and the TREX component UAP56, which is a DEAD box helicase/ATPase, in assembly of the TREX complex will also be determined. In addition, the mechanism for specifically recruiting the TREX complex to spliced mRNAs and not to unspliced pre-mRNAs will be elucidated. In Specific Aim 3, the mechanism and factors involved in export of mRNAs derived from genes that naturally lack introns will be determined. At present, little is know about this export pathway. A newly established mammalian system for assaying export of intronless mRNAs will be combined with biochemical studies to define the cis-acting sequences involved in mRNA export and the factors that function in this process. Narrative The steps in gene expression are coupled to one another via an extensive network of physical and functional interactions. The major goal of this project is to achieve a detailed understanding of the mechanisms and factors involved in the functional coupling between transcription, splicing and mRNA export.

项目摘要 蛋白质编码基因的表达是一个从RNA聚合酶II转录开始的多步骤过程 以及在细胞核中进行RNA加工，然后将成熟的mRNA输出到细胞质进行翻译。 基因表达的所有步骤都通过一个广泛的物理和功能网络进行协调 执行路径每一步的机器之间的相互作用。基因表达缺陷 是人类疾病的主要原因，相反，基因表达是主要的细胞 可以被利用来诊断和治疗疾病的过程。因此，对基因的详细了解 表达对于理解和治疗疾病都是必不可少的。该计划的长远目标 拟议的研究是为了详细了解转录与转录偶联的机制。 剪接和将剪接耦合到信使核糖核酸的输出。此外，所需的机制和因素 出口来自天然缺乏内含子的基因的mRNAs是一个核心目标。在具体目标1中，a 将建立耦合的RNAP II转录/剪接系统以与RNA结合使用 干扰以识别在偶联中起作用的蛋白质。癌蛋白TLS/FUS是一个强有力的候选基因 将被测试的蛋白质。该系统还将用于测试一种新型号，U1/SR冲压型号， 这表明U1SnRNP和SR蛋白的共转录募集到5？剪接位点 功能，以实现拼接所需的极高保真度。一种固定式联轴器 转录/剪接实验也将被建立，以研究在 共转录RNA加工。最后，最近令人兴奋的发现转录延长 将调查与U2特别相关的因素SnRNP，以确定是否添加U2 剪接体组装过程中SnRNP到前mRNA的作用与转录延伸有关。具体而言 目的2.结合生物化学方法研究mrna输出与剪接的偶联机制。 一种强大的哺乳动物细胞信使核糖核酸输出检测系统的研究。高度自动化的功能 保守的信使核糖核酸输出机制(TREX复合体)将使用该系统确定。的作用 ATP和TREX组件UAP56，它是一个死盒螺旋酶/ATPase，在TREX的组装中 复杂性也将被确定。此外，专门招募TREX复合体的机制 将阐明剪接的mRNAs和不剪接的前mRNAs。在具体目标3中，机制和 来自天然缺乏内含子的基因的mRNAs出口所涉及的因素将被确定。在… 目前，人们对这一出口途径知之甚少。一种新建立的哺乳动物检测系统 无内含子mRNAs的输出将与生化研究相结合，以确定顺式作用序列 参与了信使核糖核酸的输出以及在这一过程中起作用的因素。叙述性 基因表达的各个步骤通过一个广泛的物理和功能网络相互连接 互动。这个项目的主要目标是实现对机制的详细了解和 参与转录、剪接和mRNA输出之间的功能偶联的因子。