Schroeder KE and Yost HJ (1996), Xenopus poly (A) binding protein maternal RNA i...

XB-ART-18735

Dev Genet 1996 Jan 01;193:268-76. doi: 10.1002/(SICI)1520-6408(1996)19:3<268::AID-DVG10>3.0.CO;2-W.

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Xenopus poly (A) binding protein maternal RNA is localized during oogenesis and associated with large complexes in blastula.

Schroeder KE , Yost HJ .

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Maternal mRNAs are synthesized during oogenesis and often stored for use during early embryogenesis, before the onset of zygotic transcription. The temporal and spatial regulation of maternal RNAs is likely to be crucial mechanism for the establishment of the body pattern. In the course of a study that identified a Xenopus maternal mRNA that is translationally regulated along the dorsoventral axis, several RNAs were found to behave anomalously in polysomal analysis and are further characterized here. As controls for polysome analysis, elF4E RNA and D7.1 RNA were equally translated in both dorsal and ventral cells, whereas the cell-cell signaling factor noggin RNA was not translated in either cell type. Maternal RNAs encoding poly (A) binding protein (PABP), Vg1 and Xcat-2 were associated with large complexes that, in contrast to polysomes, were not dissociated in magnesium-free buffer. Vg1 and Xcat-2 maternal mRNAs have been shown to be localized during oogenesis to the vegetal hemisphere of the oocyte [Rebagliati et al., 1985; Mosquera et al., 1993]. In situ hybridization analysis indicated that PABP RNA was also localized during oogenesis, to the animal hemisphere in stage VI oocytes. This suggests that association of maternal mRNAs with large EDTA-insensitive mRNP complexes is correlated with intracellular localization, but the specific localization within the oocyte is dependent upon the RNA species.

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Species referenced: Xenopus laevis
Genes referenced: eif4e gdf1 nog pabpc1 pabpc4

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	Fig. 1. Translated and untranslated RNAs in 64-cell embryos. Translation initiation factors eIF4E RNA and D7.1 RNA were associated with polysomes in both dorsal and ventral cells. EF1-alpha RNA and Noggin RNA were found predominantly in nonpolysomal fractions. A. Polysomes were purified from cell lysates in the presence of eyeloheximide. RNAa were isolated from polysomal and nonpolysoma1 fractions from dorsal and ventral cells, were reverse transcribed with oligo dTo,, and subjected to FCR. Lane 1, dorsal polysomal pellet, lane 2, ventral polysomal pellet, lane 3, dorsal nonpolysomal supernatant, lane 4, ventral nonpolysomal supernatant. B. EDTA was substituted for cycloheximide in parallel preparations to dissociate translated RNAs from polysomes. Lane 5, dorsal pellet, lane 6, ventral pellet, lane 7, dorsal supernatant, lane 8, ventral supernatant. Translated RNAs that were in pellets from cycloheximide-treated lysates (A) as a result of their incorporation in polysomes were chased into the supernatants in EDTA-treated lysates (B) as a result of EDTA disruption of polysomes and release of messages. C. Noggin maternal mRNAs are not translated in dorsal and ventral cells from 64-cell embryos. Lanes are as in A.
	Fig. 2. Poly (A) Binding protein (PABP) RNA, Xcat-2 RNA and Vgl RNA are associated with large EDTA-insensitive complexes. A. RNAs isolated from polysomal and non-polysomal fractions from dorsal and ventral cells were reverse transcribed with oligo dT (15) and subjected to PCR as described in Figure 1. Lane 1, dorsal polysoma1 pellet, lane 2, ventral polysomal pellet, lane 3, dorsal nonpolysoma1 supernatant, lane 4, ventral nonpolysomal supernatant. B. In contrast to translationally active RNAs for D7.1 and eIF4E (shown in Fig. 11, Xcat-2 and PABP RNAs were pelleted in high-speed centrifugation of lysates in which EDTA was substituted for cycioheximide. This indicated that these RNAs were associated with large nonpolysomal complexes. Lane 5, dorsal pellet, lane 6, ventral pellet, lane 7, dorsal supernatant, lane 8, ventral supernatant. C. Vgl RNA asaociated with polysomal pellets in cycloheximide-treated lysates. Lane 1, dorsal polysomal pellet, lane 2, ventral polysomal pellet, lane 3, dorsal nonpolysomal supernatant, lane 4, ventral nonpolysomal supernatant. D. Pelleting of Vgl RNA in EDTA-treated lysates was variable; in some embryo preparations, Vgl behaved as a translated RNA and was in EDTA supernatants (row 1). In other preparations, Vgl remained associated with EDTA-insensitive large complexes (row 2). Lane 5, dorsal pellet, lane 6, ventral pellet, lane 7, dorsal supernatant, lane 8, ventral supernatant.
	Fig. 3, Developmental expression pattern of Xenopus PABP RNA. A. RNAs were extracted from embryos at each of the stages indicated. Stages 3, 5, and 7 are blastula before the initiation of zygotic transcription at the midblastula transition (MBT), confirming that PABP RNA is maternal. Stage 10 is early gastrula, stage 12 is late gastrula, stage 15 is early neural fold, and stage 22 is early tailbud. Ten pg of total RNA from each stage was hybridized on a northern blot with a probe for the PABP RNA. One major band of -2.5 kB was detected both in maternal and zygotic samples. An additional minor band of 1.4 kB were detected after gastrulation. B. As a control for RNA loading, the northern blot was stripped and reprobed with Xenopus ribosomal RNA probe, Xlr14.
	Fig. 4. PABP RNA is localized to the animal hemisphere in stage VI oocytes. A. Antisense probe for PABP RNA was used on various stages of Xenopus oocytes. Staining in oocytes appears to be mostly cytoplasmic in very early stages and concentrated in a ring surrounding the germinal vesicle at intermediate stages of oogenesis. B. Oocytes hybridized with a control sense probe show no staining. C. Overstaining of a stage VI oocytes with antisense PABP probe demonstrates that PABP RNA is localized to the animal hemisphere. Animal localization was determined by the location of the germinal vesicle, indicated by the open arrow. D. Stage VI oocyte, hybridized with a control sense probe and stained under conditions identical to those in C, shows no staining.
	Fig. 6. Sections of stage VI oocytes hybridized to antisense PABP RNA indicate that the RNA is localized to a thin rim beneath the stage VI oocyte cortex in the animal hemisphere. A. Section of a stage VI oocyte that was hybridized with PABP antisense probe. Boxes indicate the two areas that are enlarged in B and C. B. Enlarged region of the animal hemisphere. PABP RNA is located in a band that polyadenylais -40 pM thick, just below the outer cortex. C. Enlarged region of the vegetal cortex; no staining for PABP RNA is evident. D. PABP RNA is not localized in stage I oocytes but instead is found distributed throughout the oocyte cytoplasm. E. Enlarged region from D. Sections are 20 pM, bar = 50 +M.