mRNA splicing in early mammalian embryos
The overall aim of the experiments presented in this thesis is to investigate the onset and regulation of mRNA splicing in early mammalian development. To investigate splicing efficiency in single mouse embryos, we have constructed splicing probes that exploit the properties of fluorescent proteins. A DsRed Express-intron-EGFP construct would report splicing by the expression of the fusion DsRed-EGFP protein, when injected in mouse embryos. In the absence of splicing the vector yields only DsRed. The ability of the probe to monitor splicing was confirmed by compromising the Ul small nuclear ribonucleoprotein or by mutation of the intron's 5' splice site. Furthermore, I show that inhibiting Ul-mediated splicing at the onset of development inhibits development beyond the 2-cell stage. Following the characterization of the splicing probe, the development of nuclear speckles was investigated during early mouse development by determining the nuclear localisation of the splicing factor SC35. These experiments showed a developmental change in nuclear organisation during oocyte maturation and through embryo development. We could also show that splicing takes place as early as the 1 -cell stage embryos in the absence of nuclear speckles. Finally, the role of an SR protein, SRp38, was also investigated. SRp38 has been reported to be a potent splicing inhibitor when dephosphorylated in mitotic cells or under heat stress. SRp38 was present during all stages of the early mouse development. Oocytes and embryos were found to be sensitive to the levels of SRp38. Expression of SRp38-EGFP or depletion of SRp38 using morpholinos caused an arrest of oocytes at the GV stage and embryos at the 2-cell stage. The splicing reporter was used to reveal that these effects were independent of splicing suggesting a novel role for SRp38 in cell biology. These studies provide a novel approach to monitoring splicing activity in single living cells and that splicing activity is necessary for the onset of early mammalian development.