mRNA localization in Xenopus oocytes
Date of Award
Doctor of Philosophy (Ph.D.)
Cell Biology and Anatomy
First Committee Member
Mary Lou King, Committee Chair
The mechanism that directs Xcat-2 to the vegetal cortex of Xenopus stage IV oocytes as well as that directs Xcat-2 to the mitochondrial cloud in stage I oocytes, were characterized. The results show that although Xcat-2 normally localizes as part of the mitochondrial cloud in previtellogenic oocytes, it can localize independently of that structure at later stages and does so in a pattern identical to Vg1. RNA sequences required and sufficient to direct Xcat-2 to the vegetal cortex were mapped to a 150-nucleotide element in the $3\sp\prime$-untranslated region by injecting deletion mutant transcripts into stage IV oocytes and monitoring localization by RNase protection and autoradiography. Depolymerization of microtubules by cold-treatment prevented translocation of Xcat-2 transcripts, but did not result in the disruption or redistribution of transport particles or cortical Xcat-2 RNA. These studies suggest that Vg1 localization pathway can be divided into four steps: translocation to the perinuclear region, assemblage into transport particles on the basal side of germinal vesicle, active translocation of the particle to the vegetal subcortex, and anchoring in the vegetal cortex proper. I postulate that, by restricting the site of particle assembly and the type of microtubule motor, specific RNAs are directed to the vegetal cortex in stage IV oocytes.Following microinjection into stage I oocytes, synthetic Xcat-2 RNAs localize to the mitochondrial cloud. The Xcat-2 $3\sp\prime$UTR was found to be both required and sufficient for mitochondrial cloud localization. Further deletion analysis narrowed this localization signal to a 250-nt region at the proximal end of the $3\sp\prime$UTR. This region includes a smaller domain shown to be sufficient to direct Xcat-2 to the vegetal cortex in stage IV oocytes. In contrast to Vg1 which is actively transported to the vegetal cortex in stage IV oocytes, Xcat-2 localization to the mitochondrial cloud does not require intact microtubules. Examination of early stage I oocytes reveals a time when Xcat-2 is uniformly distributed, arguing against vectorial nuclear export into the mitochondrial cloud. The hypothesized mechanism for Xcat-2 localization to the mitochondrial cloud is that there are specific anchoring sites within the cloud that trap Xcat-2 to this location.
Zhou, Yi, "mRNA localization in Xenopus oocytes" (1995). Dissertations from ProQuest. 3387.