Functional Coupling of Steps in Genes Expression

描述（由申请人提供）： 蛋白质编码基因的表达是一个多步骤的过程，其开始于核中的转录和RNA加工（加帽、剪接和3 '端形成），然后将成熟mRNA输出到细胞质中进行翻译。基因表达中的所有步骤都是通过一个广泛的物理和功能相互作用网络相互耦合的，这一网络仍有待理解。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人类疾病的主要细胞过程之一。因此，实现对基因表达途径的详细理解至关重要。这个提议的主要目标是确定转录、剪接和mRNA输出机制之间的耦合因素和机制。Specific Aim 1的中心目标是确定转录如何与剪接偶联。目前的模型提出，早期剪接因子（U1 snRNP和SR蛋白）共转录招募到前mRNA启动剪接体组装。与U1 snRNP、SR蛋白和RNA聚合酶II相关的蛋白质FUS被鉴定为将转录与剪接偶联的强有力的候选者。FUS还与SMN复合物相关，SMN复合物在snRNP生物发生中起作用。RNAi和生物化学工作将进行，以确定FUS在耦合转录剪接和U1 snRNP生物合成中的作用。在具体目标2中，计划主要努力确定FUS突变如何导致致命的运动神经元疾病肌萎缩性侧索硬化症（ALS）。含SMN的核Gems在FUS敲低的HeLa细胞和具有FUS突变的ALS患者成纤维细胞中是缺乏的。计划开展工作以确定Gem缺乏症是否是ALS的一般表型。剪接和snRNP生物发生测定也将使用从FUS敲低的HeLa细胞制备的核提取物和含有具有ALS-引起的患者突变的FUS的核提取物进行。这些试验也将使用从含有导致人ALS的患者突变的小鼠胚胎干细胞分化的运动神经元制备的提取物进行。这项工作有望为ALS的发病机制提供有价值的见解，并可能导致这种疾病的诊断工具。最后，具体目标3的中心目标是了解TREX复合物的功能，这是一种高度保守的机制，在mRNA输出中发挥作用。TREX与剪接体Prp19复合体相连，计划确定这些复合体在天然无内含子和剪接mRNA的输出中如何发挥作用。将使用RNAi、因子募集测定和RNA-蛋白质相互作用研究的组合来确定这些复合物如何起作用。从这些研究中预期对mRNA输出的基本细胞过程的重要见解。

DESCRIPTION (provided by applicant): Summary The expression of protein-coding genes is a multi-step process that begins with transcription and RNA processing (capping, splicing and 3' end formation) in the nucleus followed by export of mature mRNA to the cytoplasm for translation. All of the steps in gene expression are coupled to one another via an extensive network of physical and functional interactions that remains to be understood. Defects in gene expression are a major cause of human diseases, and conversely, gene expression is one of the major cellular processes that can be harnessed to diagnose and treat human diseases. Thus, it is vital to achieve a detailed understanding of the gene expression pathway. The broad goal of this proposal is to identify the factors and determine the mechanisms for coupling among the transcription, splicing, and mRNA export machineries. The central goal of Specific Aim 1 is to determine how transcription is coupled to splicing. A current model proposes that early splicing factors (U1 snRNP and SR proteins) are co-transcriptionally recruited to pre-mRNA to initiate spliceosome assembly. The protein FUS, which associates with U1 snRNP, SR proteins, and RNA polymerase II, was identified as a strong candidate for coupling transcription to splicing. FUS also associates with the SMN complex, which functions in snRNP biogenesis. RNAi and biochemical work will be carried out to determine the roles of FUS in coupling transcription to splicing and in U1 snRNP biogenesis. In Specific Aim 2, a major effort is planned to determine how mutations in FUS cause the fatal motor neuron disease amyotrophic lateral sclerosis (ALS). SMN-containing nuclear Gems are deficient in FUS knockdown HeLa cells and in ALS patient fibroblasts with a FUS mutation. Work is planned to determine whether Gem deficiency is a general phenotype in ALS. Splicing and snRNP biogenesis assays will also be carried out using nuclear extracts prepared from FUS knockdown HeLa cells and nuclear extracts containing FUS with ALS- causing patient mutations. These assays will also be performed using extracts prepared from motor neurons differentiated from mouse embryonic stem cells containing human ALS-causing patient mutations. This work is anticipated to add valuable insights into the pathogenesis of ALS as well as potentially leading to diagnostic tools for this disease. Finally, the central goal of Specific Aim 3 is to understand the function of the TREX complex, which is a highly conserved machinery that functions in mRNA export. TREX is linked to the spliceosomal Prp19 complex, and work is planned to determine how these complexes function in the export of both naturally intronless and spliced mRNAs. A combination of RNAi, factor recruitment assays, and RNA- protein interaction studies will be used to determine how these complexes function. Important insights into the essential cellular process of mRNA export is anticipated from these studies.