Kloc M et al. (2000), The targeting of Xcat2 mRNA to the germinal gra...

XB-ART-11699

Dev Biol 2000 Jan 15;2172:221-9. doi: 10.1006/dbio.1999.9554.

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The targeting of Xcat2 mRNA to the germinal granules depends on a cis-acting germinal granule localization element within the 3'UTR.

Kloc M , Bilinski S , Pui-Yee Chan A , Etkin LD .

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The germ cell lineage is specified by the germ plasm, which in Xenopus laevis contains putative determinants called germinal granules. The pathway through which these structures form and how their components are assembled remain unclear. Using a combination of electron microscopy and in situ hybridization with the germinal granule-associated Xcat2 mRNA we demonstrated that the granules were derived from a branching network of granulofibrillar material within the mitochondrial cloud. Targeting of Xcat2 mRNA to the germinal granules depended on a 164-nt 3'UTR germinal granule localization element (GGLE; nt 631-795) that was distinct from the previously defined mitochondrial cloud localization element (MCLE; nt 403-630; Y. Zhou and M. L. King, 1996, Development 122, 2947-2953). This demonstrated that the Xcat 3'UTR contains a compound localization element consisting of a general element (MCLE) targeting the RNA to the mitochondrial cloud and a second element (GGLE) responsible for targeting to the germinal granules within the cloud. The GGLE when fused to Xlsirt RNA was sufficient to target this nongranule mitochondrial cloud-associated RNA to the germinal granules. This is the first example of a localization element involved in targeting an mRNA to a specific subcellular target such as the germinal granules and suggests that cis-acting elements on RNAs play an important role in the assembly of germinal granules and, therefore, the establishment of the germ cell lineage.

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Species referenced: Xenopus laevis
Genes referenced: cat2 gdf1 gfm1 nanos1 ran tbx2

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	FIG. 1. Ultrastructural analysis of germinal granule morphogenesis and Xcat2 mRNA localization during oogenesis. EM in situ hybridization was performed as described previously (Kloc et al., 1998). (A) Analysis of postnest stage 1 oocyte showing mitochondria separated by the mitochondrial cement. Xcat2 mRNA was located between the mitochondria but not within the cement (arrows). Bar, 200 nm. (B) Xcat2 mRNA is detected on branching GFM (arrows) that are associated with structures resembling forming germinal granules. Bar, 200 nm. (C) The branching GFM alongside newly formed germinal granules containing Xcat2 mRNA. These are classified as newly formed based on their association with branching GFM. Open arrows point to granules, closed arrows point to GFM. Bar, 200 nm. (D) Newly formed germinal granules with GFM still associated and containing Xcat2 mRNA. Open arrows point to germinal granules and solid arrows point to GFM. Bar, 200 nm. (E) Newly formed germinal granule with Xcat2 at surface. Bar, 50 nm. (F) Low-magnification view of germ plasm region of late stage 1 oocyte. Bar, 3 mm. Open arrows point to the germinal granules containing Xcat2 mRNA. Oocytes shown in B–E are prestage 1.
	FIG. 2. Constructs used to make synthetic RNAs to identify localization elements and summary of their ability to localize to the mitochondrial cloud (cloud) and germinal granules (granule). (A) Xcat-FL is the full-length Xcat2 mRNA, Xcat D403-630 has the MCLE deleted from the 39UTR, Xcat D631-795 has the GGLE sequence deleted, and Xlsirt contains three full sirt repeat units with a short piece of the associated unique sequence (Kloc et al., 1993). Xlscat631-795 contains the Xlsirt repeats onto which the 164-nt GGLE has been cloned at the 39end. 1 indicates ability to localize to mitochondrial cloud or to germinal granules; 2 indicates no localization to cloud or granules. A minimum of 50 oocytes were analyzed for each construct. (B) Stability assay for injected constructs. 200 pg of digoxigenin-labeled RNA was injected into oocytes after which they were cultured for 2 days, and RNA was extracted from 50 oocytes. The isolated RNA was separated by denaturing gel electrophoresis, blotted onto Genescreen Plus membrane, incubated with anti-digoxigenin alkaline phosphatase antibody, and analyzed by color reaction. Shown is a composite of several different blots with the bands aligned. In reality each species of RNA ran at a different mobility.
	FIG. 3. Analysis of the localization of injected Xcat2 and Xlsirt RNAs. (A) Whole mount of a late stage 1 oocyte injected with 200 pg of full-length Xcat2 mRNA that was labeled with digoxigenin-11–UTP. Injected oocytes were incubated for 2 days and the RNA was detected by incubation with anti-digoxigenin alkaline phosphatase-conjugated antibody followed by a color reaction according to Kloc and Etkin (1999). Bar, 100 mm. (B) EM analysis of germ plasm region of mitochondrial cloud showing silver-enhanced gold particles associated with the germinal granules. Bar, 100 nm. (C) Whole-mount analysis of late stage 1 oocyte injected with 200 pg of Xlsirt RNA. Bar, 100 mm. (D) EM analysis of germ plasm region of mitochondrial cloud showing Xlsirt RNA associated with fibrous network. Bar, 100 nm. (E) Analysis of Vg1 mRNA localization in stage 1 oocyte showing low level of background labeling in cloud. Bar, 250 nm. Solid arrows point to either the cloud (A and C) or the silver-enhanced gold particles (B, D, and E). Open arrows point to germinal granules.
	FIG. 4. Analysis of the localization of the RNA from the Xcat D403-630 and Xlscat631-795 constructs. (A) Whole-mount analysis of the localization of injected Xcat D631-795 RNA to the mitochondrial cloud. Bar, 100 mm. (B) EM analysis showing that this RNA did not target the germinal granules. Bar, 100 nm. (C) Whole-mount in situ hybridization analysis of oocyte injected with Xcat D403-630, which does not show localization to the cloud. (D) Whole-mount analysis of injected RNA from the Xlscat631-795 construct showing localization to the cloud. Bar, 100 mm. (E and F) EM analysis showing that the GGLE associated with Xlsirts in the Xlscat631-795 construct targets the Xlsirts to the germinal granules. Solid arrows point to either the cloud (A and D) or the silver-enhanced gold particles (B, E, and F). Open arrows point to germinal granules. Bar, 100 nm.
	FIG. 5. Summary of germinal granule formation. Xcat2 mRNA was detected at the periphery of the mitochondrial cement (red) in oocytes just after being surrounded by follicle cells. Mitochondria (green) are embedded within the cement (Post nest). The next phase involves association of Xcat2 mRNA with the newly formed GFM (red) (Early prestage 1). At early stage 1 we see the aggregation of the GFM into small germinal granule-like structures. By stages 1 and 2 we detect the Xcat2 mRNA associated with the fully formed germinal granules. The association of Xcat2 mRNA with these structures involves the presence of a cis-acting element that is different from the element needed for association with the cloud.