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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.
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 oocytecytoplasm. E. Enlarged region from D. Sections
are 20 pM, bar = 50 +M.
