THE LINK BETWEEN SPLICING & EXPORT OF MESSENGER RNA

基因表达是一个多步骤的过程，从细胞核前mRNA的合成开始，到细胞质中mRNA的翻译结束。在这两个事件之间，pre- mrna经历了几个加工步骤，包括盖帽、剪接和3'端形成。与基因表达的其他步骤相比，我们对后生动物的mRNA输出知之甚少。本研究的重点是利用非洲爪蟾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。第一个目标是确定导致有效mRNA输出的前mRNA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家族的必要剪接因子）的作用将被研究。在第二个目标中，一种新的复合物在体外组装在剪接的mRNA上，并有效地运输出卵母细胞核，将被表征。这一目标将通过建立一种分离剪接mRNA复合体的方法来实现。将注射到卵母细胞核中，以确定纯化的复合物是否具有功能，并对其蛋白质组成进行详细分析。该复合物要么是mRNA输出底物的处理器，要么本身就是输出底物，因此其表征将为mRNA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，第四个目标侧重于识别在mRNA输出中起作用的接头和/或受体。该策略是鉴定介导基本输出因子RanGTP与输出底物结合的蛋白质。总之，这些研究应该为mRNA输出的机制以及剪接与输出之间的联系提供重要的新见解。

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.