Functional Coupling of Steps in Gene Expression

描述（由申请人提供）：以及在细胞核中进行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网络来协调的，这个网络由执行途径每个步骤的机器之间的物理和功能相互作用组成。基因表达缺陷是人类疾病的主要原因，相反，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至关重要。拟议的研究的长期目标是实现一个详细的了解耦合转录剪接和耦合剪接mRNA输出的机制。此外，从天然缺乏内含子的基因导出mRNA所需的机制和因素是一个中心目标。在具体目标1中，将建立一个耦合RNAP II转录/剪接系统，与RNA干扰结合使用，以鉴定在耦合中起作用的蛋白质。癌蛋白TLS/FUS是一个强有力的候选蛋白，将进行测试。该系统还将用于测试一种新的模型，即U1/SR冲压模型，该模型提出U1 snRNP和SR蛋白的共转录募集到5'剪接位点的功能，以实现剪接所需的极高保真度。还将建立固定化偶联转录/剪接测定，以研究在共转录RNA加工中起作用的机制。最后，最近令人兴奋的发现，转录延伸因子是专门与U2 snRNP将进行调查，以确定是否添加U2 snRNP的前mRNA剪接体组装过程中耦合到转录延伸。在具体目标2中，将通过将生物化学研究与用于测定哺乳动物细胞中mRNA输出的强大系统相结合来研究mRNA输出与剪接的耦合机制。将使用该系统确定高度保守的mRNA输出机制（TREX复合物）的功能。还将确定ATP和TREX组分UAP 56（其是DEAD盒解旋酶/ATP酶）在TREX复合物组装中的作用。此外，将阐明TREX复合物特异性募集至剪接mRNA而非未剪接前体mRNA的机制。在特定目标3中，将确定来自天然缺乏内含子的基因的mRNA的输出所涉及的机制和因素。目前，人们对这一出口途径知之甚少。一个新建立的哺乳动物无内含子mRNA的出口检测系统将与生物化学研究相结合，以确定参与mRNA出口的顺式作用序列和在这一过程中发挥作用的因素。

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.