THE LINK BETWEEN SPLICING & EXPORT OF MESSENGER RNA

拼接之间的联系

• 批准号: 6627247
• 负责人: ROBIN E. REED
• 金额: $ 28.34万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-01-01 至 2003-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6627247
• 关键词: RNA binding protein; RNA splicing; Xenopus; Xenopus oocyte; gene expression; intracellular transport; messenger RNA; microinjections; nucleic acid sequence

Gene expression is a multi-step process that begins with synthesis of pre-mRNA in the nucleus and eliminates with translation of mRNA in the cytoplasm. Between these two events, pre-mRNAs undergo several processing steps, including capping, splicing, and 3' end formation. In contrast to the other steps in gene expression, very little is known about mRNA export in metazoans. This proposal is focused on using the Xenopus oocyte microinjection system to investigate the recently detected functional link between the splicing and mRNA export pathways. The first aim is to identify the important sequence and organizational features of pre-mRNA that lead to efficient mRNA export. In particular, the role of exon sequences and their binding factors (the SR family of essential splicing factors) will be investigated. In the second aim, a novel complex that assembles on spliced mRNA in vitro, and is efficiently transported out of oocyte nuclei, will be characterized. This goal will be achieved by establishing a method for isolating the spliced mRNA complex. Injection into oocyte nuclei will be used to determine whether the purified complex is functional, and detailed analysis of its protein composition will be carried out. This complex is either a processor to the mRNA export substrate or is itself the export substrate, and thus its characterization will provide valuable insights into the mRNA export pathway. In the third aim, studies will be carried out to determine whether the coupling between splicing and export may serve as a checkpoint for detecting splicing mutations. Finally, the fourth aim is focused on identifying adaptors and/or receptors that function in mRNA export. The strategy is to identify proteins that mediate binding of the essential export factor RanGTP to export substrates. Together, these studies should provide important new insights into the mechanism of mRNA export and the link between splicing and export.

基因表达是一个多步骤的过程，它始于细胞核中前-mRNA的合成，并随着细胞质中mRNA的翻译而消失。在这两个事件之间，前mRNAs经历了几个处理步骤，包括封端、剪接和3‘端形成。与基因表达的其他步骤相比，后生动物中的信使核糖核酸输出知之甚少。这项建议的重点是使用非洲爪哇卵母细胞显微注射系统来研究最近发现的剪接和mRNA输出途径之间的功能联系。第一个目的是确定导致高效信使核糖核酸输出的前信使核糖核酸的重要序列和组织特征。特别是，外显子序列及其结合因子(SR家族的基本剪接因子)的作用将被研究。在第二个目标中，将表征一种在体外组装在剪接的mRNA上并有效地转运出卵母细胞核的新型复合体。这一目标将通过建立一种分离剪接的mRNA复合体的方法来实现。注射入卵母细胞核将用于确定纯化的复合体是否具有功能，并将对其蛋白质组成进行详细分析。这种复合体既是信使核糖核酸输出底物的加工者，也是信使核糖核酸输出底物，因此它的特性将为研究信使核糖核酸输出途径提供有价值的见解。在第三个目标中，将进行研究，以确定剪接和输出之间的耦合是否可以作为检测剪接突变的检查点。最后，第四个目标是确定在信使核糖核酸输出中起作用的接头和/或受体。我们的策略是确定介导基本出口因子RanGTP与出口底物结合的蛋白质。综上所述，这些研究应该会对mRNA输出的机制以及剪接和输出之间的联系提供重要的新见解。