Splicing promotes mRNA export in mammalian cells

Gene expression in metazoans begins with transcription of pre-mRNAs. These transcripts undergo many processing steps before they are exported to the cytoplasm and translated into protein. These steps include capping at the 5' end, splicing to remove introns, and cleavage and polyadenylation at the 3' end. Although distinct cellular machineries carry out each processing step, there is extensive physical and functional coupling among them. The physical coupling between the splicing and export machineries provides one of the few examples in which this coupling has been characterized. Specifically, studies show that the mRNA export machinery co-localizes with splicing factors in nuclear speckle domains, is loaded onto the mRNA during splicing and is recruited more efficiently to spliced mRNAs than to cDNA transcripts. Despite these findings, whether splicing promotes mRNA export remains controversial. In this dissertation, I have carried out a systematic analysis of the role of splicing in mRNA export. To do this, intron-containing genes or their corresponding cDNA counterparts were either transfected or microinjected into HeLa cell nuclei. Fluorescence in situ hybridization (FISH) was used to detect and quantitate the nucleocytoplasmic distribution of the mRNAs. These analyses indicate that both the kinetics and efficiency of mRNA export arc enhanced 3-10 fold (depending on the construct) for spliced mRNAs relative to their cDNA counterparts. This splicing-dependent enhancement of mRNA export was observed for three different genes and in two different cell types (HeLa and SV40-MEFs), indicating that the functional coupling of splicing to mRNA export is a conserved and general feature of gene expression in higher eukaryotes. Finally, consistent with previous studies, this dissertation shows that splicing leads to a significant enhancement in overall mRNA levels, and I present preliminary evidence that this enhancement may be due to a splicing-dependent nuclear surveillance mechanism.