Localization of RNAs to the Mitochondrial Cloud inXenopusOocytes through Entrapment and Association with Endoplasmic Reticulum

Abstract

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.

Differential expression of fertility genes boule and dazl in Chinese sturgeon (Acipenser sinensis), a basal fish

“…As described previously (Chang et al 2004;Kosaka et al 2007;Li et al 2011;Peng et al 2009;Xu et al 2009), dazl colocalized with a cytoplasm structure, named the Balbiani body (BB) or mitochondrial cloud (MC) (King et al 2005), containing mitochondria, endoplasmic reticulum and germ plasm RNAs. In particular, this structure could be detected by a mitochondrial marker, such as MitoTracker.…”

“…og oogonia, sg spermatogonia, psp primary spermatocytes, ssp secondary spermatocytes, spd, spermatid. Bars (a, d) 50 μm, (b, e) 20 μm shown that dazl RNA of Xenopus (Chang et al 2004), zebrafish (Kosaka et al 2007), gible carp (Peng et al 2009), medaka (Xu et al 2009) and trout (Li et al 2011) are concentrated within the BB/MC, a spherical-shaped structure. Nonetheless, in Acipenser gueldenstaedtii (Zelazowska et al 2007), oocytes contain two different zones of cytoplasm, the homogeneous ooplasm and granular ooplasm, with the granular ooplasm corresponding to the BB/MC.…”

Maternal syntabulin is required for dorsal axis formation and is a germ plasm component in Xenopus

“…In the METRO pathway, mRNAs accumulate within a region of the Balbiani body, also called the mitochondrial cloud, in stage I oocytes (Chang et al, 2004), and then translocate to the vegetal cortex through a directional expansion of the mitochondrial cloud itself (Wilk et al, 2004). Interestingly, many mRNAs localized through the METRO pathway are also components of the germ plasm and involved in germ cell differentiation (Kloc et al, 1998).…”

Genomic analysis of RNA localization

“…63,64 Second, mRNAs passively diffusing through the cytoplasm can be bound and retained by anchoring proteins that are themselves localized to specific cellular sites. This system has been shown to function in at least two different systems: Xenopus oocytes 65 with the RNAs Xcat2 and Xdazl and Drosophila embryos with nanos mRNA. 66 Finally, RNAs can also be actively directed to a particular location.…”