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Vol. 15, Issue 10, 4669-4681, October 2004

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Localization of RNAs to the Mitochondrial Cloud in Xenopus Oocytes through Entrapment and Association with Endoplasmic Reticulum

Patrick Chang ^*, Jan Torres , Raymond A. Lewis , Kimberly L. Mowry , Evelyn Houliston ^* , and Mary Lou King 

^* Unité Mixte de Recherche 7009 Centre National pour la Recherche Scientifique/Université Pierre et Marie Curie, Observatoire Océanologique, 06230 Villefranche sur Mer, France; Department of Cell Biology and Anatomy, University of Miami School of Medicine, Miami, FL 33136; and Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, RI 02912

Submitted March 29, 2004; Revised July 15, 2004; Accepted July 23, 2004
Monitoring Editor: Susan Strome

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.

Abbreviations used: PGC, primordial germ cell; FRAP, fluorescence recovery after photobleaching; MC, mitochondrial cloud; UTR, untranslated region; RBP, RNA-binding protein; LS, localization signal; GGLE, germinal granule localization element; ER, endoplasmic reticulum; BSA, bovine serum albumin; GFP, green fluorescent protein; PBS, phosphate-buffered saline; GV, germinal vesicle; LE, localization element; Dil, DilC₁₆(3); DiO, DiOC₍₆₎(3).

The online version of this article contains supplemental material accessible through http://www.molbiolcell.org

Corresponding author. E-mail address: houliston{at}obs-vlfr.fr.

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