RNA Transport to the Vegetal Cortex ofXenopusOocytes

Abstract

Xcat-2 RNA, a component of the germ plasm inXenopus,localizes with the mitochondrial cloud material to the vegetal cortex in stage II oocytes.Vg1 RNA also localizes to the vegetal cortex, but later in stage III/IV oocytes, using a microtubule dependent pathway. To further analyze the mechanisms involved in RNA transport,in situhybridization and autoradiography were used to follow the localization of endogenousVg1 and injectedXcat-2 transcripts in stage IV oocytes. We show thatXcat-2 is competent to localize to the vegetal cortex quite independently of the mitochondrial cloud.Xcat-2 RNA appears to use the lateVg1 localization pathway, as depolymerization of microtubules by cold or nocodazole treatment prevented translocation ofXcat-2 transcripts, but did not result in the disruption ofXcat-2 anchored in the cortex. Furthermore, RNA transport was shown to be stage dependent for bothVg1 andXcat-2 RNAs, as they did not localize in fully grown stage VI oocytes after injection. RNA sequences both required and sufficient to directXcat-2 to the vegetal cortex were mapped to a sequence of 150 nt immediately adjacent to the open reading frame and additional sequences at the end of the 3′ untranslated region. Mapping was accomplished by injecting deletion mutant transcripts into stage IV oocytes and monitoring localization by RNase protection and autoradiography. All mutants competent for translocation were also capable of cortical anchoring, suggesting that the same signal is used for both steps. We speculate that two separate RNA pathways evolved during the course ofXenopusoogenesis. One pathway, specialized for the transport of germ plasm by way of the mitochondrial cloud, occurs early to ensure the segregation of the germ cell lineage. The other, late, pathway may serve as the more general transport system for localizing RNAs involved in somatic cell differentiation.