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 i s 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.
