Functional Coupling of Steps in Gene Expression

描述（由申请人提供）：在细胞核中进行RNA加工，然后将成熟mRNA输出到细胞质中进行翻译。基因表达的所有步骤都是通过一个广泛的物理和功能相互作用的网络来协调的，这些网络在执行路径的每个步骤的机器之间。基因表达缺陷是人类疾病的主要原因，反过来，基因表达是可以用来诊断和治疗疾病的主要细胞过程之一。因此，对基因表达的详细了解对于理解和治疗疾病都是必不可少的。本研究的长期目标是详细了解转录与剪接的耦合机制以及剪接与mRNA输出的耦合机制。此外，从天然缺乏内含子的基因中导出mrna所需的机制和因素是一个中心目标。在Specific Aim 1中，将建立一个耦合的RNAP II转录/剪接系统，结合RNA干扰来识别具有耦合功能的蛋白质。癌蛋白TLS/FUS是一个强有力的候选蛋白，将被测试。该系统还将用于测试一个新的模型，U1/SR冲压模型，该模型提出U1 snRNP和SR蛋白在5'剪接位点的共转录招募功能，以实现剪接所需的极高保真度。为了研究共转录RNA加工的作用机制，还将建立固定化偶联转录/剪接试验。最后，我们将研究最近令人兴奋的发现，即转录延伸因子与U2 snRNP特异性相关，以确定在剪接体组装过程中将U2 snRNP添加到前mrna是否与转录延伸相耦合。在Specific Aim 2中，我们将结合生化研究和一个强大的哺乳动物细胞mRNA输出分析系统来研究mRNA输出与剪接耦合的机制。高度保守的mRNA输出机制（TREX复合体）的功能将使用该系统确定。ATP和TREX组分UAP56（一种DEAD盒解旋酶/ATP酶）在TREX复合物组装中的作用也将被确定。此外，将阐明TREX复合物特异性招募到剪接mrna而不是未剪接的pre- mrna的机制。在Specific Aim 3中，将确定天然缺乏内含子的基因衍生的mrna输出的机制和因素。目前，人们对这种出口途径知之甚少。一个新建立的用于分析无内含子mRNA输出的哺乳动物系统将与生化研究相结合，以确定mRNA输出中涉及的顺式作用序列和在此过程中起作用的因素。

DESCRIPTION (provided by applicant): 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.