Localization of RNAs to the mitochondrial cloud in Xenopus oocytes through entrapment and association with endoplasmic reticulum.
The germ cell lineage in Xenopus is specified by the inheritance of germ plasm, which originates within a distinct "mitochondrial cloud" (MC) in previtellogenic oocytes. Germ plasm contains localized RNAs implicated in germ cell development, including Xcat2 and Xdazl. To understand the mechanism of the early pathway through which RNAs localize to the MC, we applied live confocal imaging and photobleaching analysis to oocytes microinjected with fluorescent Xcat2 and Xdazl RNA constructs. These RNAs dispersed evenly throughout the cytoplasm through diffusion and then became progressively immobilized and formed aggregates in the MC. Entrapment in the MC was not prevented by microtubule disruption and did not require localization to germinal granules. Immobilized RNA constructs codistributed and showed coordinated movement with densely packed endoplasmic reticulum (ER) concentrated in the MC, as revealed with Dil16(3) labeling and immunofluorescence analysis. Vg1RBP/Vera protein, which has been implicated in linking late pathway RNAs to vegetal ER, was shown to bind specifically both wild-type Xcat2 3' untranslated region and localization-defective constructs. We found endogenous Vg1RBP/Vera and Vg1RBP/Vera-green fluorescent protein to be largely excluded from the MC but subsequently to codistribute with Xcat2 and ER at the vegetal cortex. We conclude that germ line RNAs localize into the MC through a diffusion/entrapment mechanism involving Vg1RBP/Vera-independent association with ER.
Unité Mixte de Recherche 7009 Centre National pour la Recherche Scientifique/Université Pierre et Marie Curie, Observatoire Océanologique, 06230 Villefranche sur Mer, France.
Patrick
P
Chang
Jan
J
Torres
Raymond A
RA
Lewis
Kimberly L
KL
Mowry
Evelyn
E
Houliston
Mary Lou
ML
King
D000818:Animals; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D036681:Fluorescence Recovery After Photobleaching; D005456:Fluorescent Dyes / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism*; D009865:Oocytes / Q000166:cytology / Q000502:physiology*; D012313:RNA / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins; D029867:Xenopus Proteins / Q000235:genetics / Q000378:metabolism; D014982:Xenopus laevis
D005456:Fluorescent Dyes; C498635:IGF2BP3 protein, Xenopus; D016601:RNA-Binding Proteins; D012097:Repressor Proteins; D029867:Xenopus Proteins; C558784:nanos1 protein, Xenopus; D012313:RNA