Coupling Transcription, Splicing and mRNA Export

偶联转录、剪接和 mRNA 输出

• 批准号: 6897895
• 负责人: ROBIN E. REED
• 金额: $ 97.96万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6897895
• 关键词: DNA directed RNA polymerase; RNA splicing; Xenopus oocyte; gene mutation; genetic transcription; helicase; intermolecular interaction; laboratory rabbit; messenger RNA; polymerase chain reaction; protein protein interaction; protein structure function; spliceosomes

DESCRIPTION (provided by applicant): Expression of protein-coding genes is a multi-step process beginning with transcription by RNA polymerase II in the nucleus. During transcription, the nascent pre-mRNA undergoes several processing steps including capping, splicing, and polyadenylation. The mature mRNA is then exported to the cytoplasm for translation. Although each of the steps in gene expression is carried out by a distinct cellular machine, growing evidence indicates that there is an extensive network of coupled interactions between each machine. At present, however, little is understood about the mechanisms involved in the coupling. The goal of this proposal is to determine the mechanisms for coupling transcription, splicing, and mRNA export. In Specific Aim 1, a functional assay will be used to determine whether transcription by RNA polymerase II promotes mRNA export. In addition, the recently identified TREX complex, which may function in coupling all three processes, will be investigated in detail. All of the full-length human TREX components will be cloned, expressed, and antibodies generated. These reagents will be used to test the function of the TREX complex and to determine how this complex associates with active genes and their transcripts. In Specific Aim 2, the mechanism for coupling transcription to splicing will be investigated using an in vitro system for coupling transcription by RNA polymerase II to splicing. Studies will also be carried out to determine whether splicing factors, including U1 and U2 snRNPs, are recruited to actively transcribed genes. Finally, in Specific Aim 3, coupling of splicing to mRNA export will be investigated by isolating and determining the composition of the spliced mRNP that promotes export. In addition, the role of the conserved DEAD box helicase protein UAP56, which functions in export and is also present in both the TREX complex and spliceosome, will be determined using specific mutations in this protein combined with functional assays for coupling.

描述(申请人提供)：蛋白质编码基因的表达是一个多步骤的过程，从细胞核中的RNA聚合酶II转录开始。在转录过程中，新生的前信使核糖核酸经历了几个处理步骤，包括封端、剪接和聚腺苷酸化。然后，成熟的mRNA被输出到细胞质中进行翻译。尽管基因表达的每一步都是由不同的细胞机器执行的，但越来越多的证据表明，每台机器之间存在着广泛的耦合相互作用网络。然而，目前对这种耦合所涉及的机制知之甚少。这个建议的目标是确定耦合转录、剪接和信使核糖核酸输出的机制。在特定的目标1中，将使用功能分析来确定RNA聚合酶II的转录是否促进了mRNA的输出。此外，将对最近发现的可能连接所有三个过程的TREX复合体进行详细调查。所有全长的人类TREX组件都将被克隆、表达并产生抗体。这些试剂将用于测试TREX复合体的功能，并确定该复合体如何与活性基因及其转录本相关联。在特定目标2中，将使用RNA聚合酶II将转录与剪接耦合的体外系统来研究转录与剪接的耦合机制。还将进行研究，以确定包括U1和U2 SnRNPs在内的剪接因子是否被招募到活跃转录的基因中。最后，在具体目标3中，将通过分离和确定促进出口的剪接mRNP的组成来研究剪接与mRNA输出的耦合。此外，保守的死盒解旋酶蛋白UAP56在出口中发挥作用，也存在于TREX复合体和剪接体中，将使用该蛋白的特定突变结合偶联功能分析来确定该蛋白的作用。