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Xcat-2 RNA, a component of the germ plasm in Xenopus, localizes with the mitochondrial cloud material to the vegetal cortex in stage II oocytes. Vg1 RNA also localizes to the vegetal cortex, but later in stage III/IV oocytes, using a microtubule dependent pathway. To further analyze the mechanisms involved in RNA transport, in situ hybridization and autoradiography were used to follow the localization of endogenous Vg1 and injected Xcat-2 transcripts in stage IV oocytes. We show that Xcat-2 is competent to localize to the vegetal cortex quite independently of the mitochondrial cloud. Xcat-2 RNA appears to use the late Vg1 localization pathway, as depolymerization of microtubules by cold or nocodazole treatment prevented translocation of Xcat-2 transcripts, but did not result in the disruption of Xcat-2 anchored in the cortex. Furthermore, RNA transport was shown to be stage dependent for both Vg1 and Xcat-2 RNAs, as they did not localize in fully grown stage VI oocytes after injection. RNA sequences both required and sufficient to direct Xcat-2 to the vegetal cortex were mapped to a sequence of 150 nt immediately adjacent to the open reading frame and additional sequences at the end of the 3' untranslated region. Mapping was accomplished by injecting deletion mutant transcripts into stage IV oocytes and monitoring localization by RNase protection and autoradiography. All mutants competent for translocation were also capable of cortical anchoring, suggesting that the same signal is used for both steps. We speculate that two separate RNA pathways evolved during the course of Xenopus oogenesis. One pathway, specialized for the transport of germ plasm by way of the mitochondrial cloud, occurs early to ensure the segregation of the germ cell lineage. The other, late, pathway may serve as the more general transport system for localizing RNAs involved in somatic cell differentiation.
FIG. I. Xcat-2 RNA microinjected into stage IV oocytes localizes
to the vegetal half. (A) Schematic of the construct used to
make transcripts for injection. Open box represents the 5' leader
sequence of Xenopus ,B-globin mRNA, -50 bp long. Filled box
represents the 5' leader and coding region of Xcat-2, -400 bp
long. Thick line represents the 3' UTR of Xcat-2, -410 bp long.
The region protected in an RNase protection assay and the size
of the probe are indicated. (B) The concentration of Xcat-2 in
animal (A) or vegetal (V) halves on Day 0 (DO) through Day 4
(D4) of culture was assessed by RNase protection. 100 pg of
capped Xcat-2 transcript was injected into stage IV oocytes. Total
RNA from two oocyte halves were loaded on each lane. In this
example, localization appears complete after Day 3. Endogenous
Xcat-2 served as an internal control for oocyte viability and accurate
cutting of A/V halves. ODC (ornithine decarboxylase) was
included as a control for RNA loading. Yeast tRNA served as a
control for nonspecific hybridization. The labeled probes for
ODC and Xcat-2 are shown as well as DNA markers in bp.
FIG. 2. Injected Xcat-2 mRNA uses the same localization pathway as endogenous Vg1 in stage IV oocytes. (AâD) In situ hybridizations
showing Vg1 localization using DIG antisense probes. (A) Section of a stage III oocyte. Vg1 moving toward the nucleus. (B) Section of a
stage III oocyte. Endogenous Vg1 starts to concentrate around the nucleus and moves toward one pole (the vegetal pole). (C) Section of
an early stage IV oocyte; more Vg1 appears near the vegetal cortex. Signal is very distinctive. (D) Section of late stage IV oocyte. Majority
of the endogenous Vg1 is found in the vegetal cortex. (EâH) Autoradiograms showing localization of injected 35S-labeled, capped, and
polyadenylated Xcat-2 RNA after injection into early stage IV oocytes. (E) After 1 day of culture, silver grains were observed throughout
the cytoplasm with a concentration on the basal side of the nucleus and into the vegetal half. (F) After 2 days, grains were observed
concentrated mainly on the basal side of the nucleus and within aggregates between the nucleus and the cortex. (G) After 3 days, Xcat-
2 was observed in the cortex. (H) After 4 days, the majority of the label was in the oocyte cortex, although a significant amount of Xcat-
2 remains in the vegetal yolky area. Bar in A for AâH, 130 mm.
FIG. 3. The translocation process of Xcat-2 in stage IV oocytes is
microtubule dependent and the integrity of Xcat-2 translocation
complexes is microtubule independent. Autoradiograms of IV oocytes
injected with 35S-labeled , capped, and polyadenylated Xcat-2
transcripts. (A) Oocyte cultured for 2 days and then placed on ice
for 90 min. (B) The same as in (A) but then cultured in the presence
of 1 JLgl ml of nocodazole at 20oc for 24 hr. Note that the Xcat-2
transcripts remain asymmetrically distributed and within complexes.
(C) The same as in (A) and then cultured at 20°C without
nocodazole for an additional 4 days. Virtually all of the injected
transcripts reach the vegetal cortex. (C') Another example of an
oocyte treated as in C. Bar in A for A-C', 130 JLm.
FIG. 4. The 3'UTR of Xcat-2 is required and sufficient for Xcat-
2 localization in stage IV oocytes. (A) Diagrams of the eDNA constructs
used for making injected transcripts. The probes for the
RNase protection assays are shown by thin lines with their sizes
indicated. Open boxes, the 5' leader sequence of Xenopus ,B-globin
(-50 bp). Shaded box, the 5' leader and coding sequence of Xcat-2
(-400 bp). Thick line, the 3'UTR of Xcat-2 (-410 bp). Striped box,
a portion of luciferase coding region. (B) An RNase protection assay
for localization of Xcat-2 S'ORF and 3'UTR after Day 0 (DO) and
Day 4 (D4) of culture. Total RNA from animal (A) or vegetal M
halves was assayed. The 3'UTR of Xcat-2 and the luciferase-tagged
3'UTR of Xcat-2 are localized to the vegetal half of oocytes, while
the 5'0RF/X,BG3'UTR is not. (DO) One-quarter of an oocyte equivalent
was loaded; (D4) two oocyte equivalents were loaded. ODC
probe was included as a control for RNA loading.
FIG. 5. A cis-localization signal located within the 3*UTR is necessary and sufficient for Xcat-2 mRNA localization. (A) Sections of
oocytes showing examples of injected deletion mutants that failed to localize (a), localized weakly (b), or localized strongly (c). (B) Schematic
of the series of the mutant transcripts tested for localization in stage IV oocytes. Deletions are indicated by interruptions in the lines.
After the culture period, localization of the injected transcripts was scored as negative (0) or positive (/). Failure to localize was always
absolute. Oocytes were scored as positive that ranged from what is depicted in (b) and (c). The vegetal half localizations were monitored
by RNase protection assays. Localization to the vegetal cortex was monitored by autoradiography (radiolabeled transcripts) or by in situ
hybridizations (DIG-labeled probe) (see Fig. 4A and Materials and Methods for details). (C) Histogram showing quantitation of the RNase
protection assays from two series of experiments using the PhosphoImager (Molecular Dynamics). Results are plotted for each mutant
transcript as the percentage gained or lost in the animal or vegetal halves at the end of the culture period relative to the total remaining.
FIG. 6. Injected Xcat-2 and Vg1 transcripts localize in stage IV
oocytes but not in stage VI oocytes. (A) A diagram of the eDNA
constructs used in making Xcat-2 or X,BG-340/3' transcripts.
Striped box indicates the coding region of Xenopus ,B-globin (-490
bp). Open thick line indicates the Vg1 localization element (340
bp). Thin line indicates the probe for RNase protection assays
(-350 bp). Xcat-2 construct is the same as that indicated in Fig.
1A. (B) An RNase protection assay with total RNA isolated from
animal (A) or vegetal M halves of the injected oocytes. 50-100 pg
each of Xcat-2 and X,BG/340 transcript was injected into each oocyte.
Ten oocyte halves were collected for each time point. For Day
0 (DO) one-quarter of an oocyte equivalent was loaded. For Day 4
(D4), two oocyte equivalents were loaded. ODC (ornithine decarboxylase)
probe was included as a control for RNA loading. Note
that neither injected Vg1 nor Xcat-2 transcripts localized in stage
VI oocytes.
