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The mitochondrial cloud is a unique cell structure found in stage I Xenopus oocytes that plays a role in mitochondriogenesis and in the distribution of germ plasm to the vegetal pole. Xcat-2 RNA specifically localizes to the mitochondrial cloud and moves with it to the vegetal subcortex in stage II oocytes. Later, in the 4-cell embryo, it is found in a pattern identical to the germ plasm. Following microinjection into stage I oocytes, synthetic Xcat-2 RNAs localize to the mitochondrial cloud within 22 hours. Transcripts are stable over this time period with very little evidence of degradation. The Xcat-2 3'untranslated region was found to be both required and sufficient for mitochondrial cloud localization. Further deletion analysis narrowed this localization signal to a 250 nucleotide region at the proximal end of the 3'untranslated region. This region is different from, but overlaps with, a domain previously shown to be sufficient to direct Xcat-2 to the vegetal cortex in stage IV oocytes. Examination of early stage I oocytes reveals a time when Xcat-2 is uniformly distributed, arguing against vectorial nuclear export into the mitochondrial cloud. Analysis of localization at different time points does not suggest active transport to the mitochondrial cloud. We postulate that localization occurs by selective entrapment of Xcat-2 within the cloud by localized binding sites.
Fig. 1. Xcat-2 mRNA progressively localizes to and is found exclusively in the
mitochondrial cloud of stage 1 oocytes. In situ hybridizations with stage 1 oocytes
approximately 250 mm (A-C) or 50-70 mm (D-F) in diameter. Detection was by a
digoxigenin-labeled antisense RNA probe for (A) the mitochondrial large ribosomal RNA
(LrRNA), (B,D-F) Xcat-2 and (C) a mixture of both probes. (A) The LrRNA probe
hybridizes to the mitochondrial aggregates surrounding the nucleus as well as to the large
mitochondrial cloud. (B) Endogenous Xcat-2 RNA is present only in the mitochondrial
cloud. (C) A mixture of both probes reveals that Xcat-2 and the mLrRNA co-localize
with the mitochondrial cloud as no additional structures were detected. All oocytes shown
in D-F were processed together on one slide. (D) Endogenous Xcat-2 in small stage I
oocytes is uniformly distributed in the cell. (E) Xcat-2 partially localized to the
mitochondrial cloud. (F) Mitochondrial cloud localization appears complete for Xcat-2.
Bar, 80 mm.
Fig. 2. Endogenous Xcat-2 levels remain constant throughout
oogenesis. The concentration of Xcat-2 and Vg1 in stage I, II, III, IV
early (E), IV late (L), V and VI oocytes was determined by RNase
protection. The steady state level of Xcat-2 remains the same
throughout oogenesis, arguing against RNA degradation as a
mechanism for mitochondrial cloud localization. (A) Diagram of
Xcat-2 mRNA with 5¢ leader (open box), coding region (shaded box),
3¢UTR (thick line). (B) RNase protection. Two oocyte-equivalents
were loaded per lane. The probes used were Vg1 (~400 nt), Xcat-2
(240 nt) and ODC (120 nt) as indicated. Note that Xcat-2 appears to
accumulate before Vg1 does in late stage I oocytes. The Vg1 antisense
probe is protected from RNase digestion by Vg1 transcripts to give
multiple bands as shown previously (Rabagliati et al., 1985; Melton,
1987). ODC (ornithine decarboxylase) was used as a control for RNA
loading (Bassez et al., 1990) and yeast total RNA (20 mg) was used as
a control for specificity. (C) RNase protection showing Xcat-2 is
present in young oocytes (50-70 mm; as in Fig. 1D) and increases in
amount in late stage I oocytes (250 mm) relative to the ODC control.
Fig. 3. Xcat-2 RNA, but not Vg1 RNA, contains a
signal required and sufficient for mitochondrial
cloud localization. (A-E) Oocytes were injected
with 35S-labeled Xcat-2 or XbG-340/3¢ transcripts
on the side opposite the mitochondrial cloud (A,B)
22 hours after injection, Xcat-2 is localized within
the mitochondrial cloud whereas (C) the Vg1
XbG-340/3¢ transcript remains unlocalized. (D)
The Xcat-2 5¢ORF fails to localize but the Xcat-2
3¢UTR (E) localizes as efficiently as does the fulllength
sequence (B). (F) Oocyte injected with a
DIG-labeled chimeric transcript containing the
luciferase-tagged 3¢UTR of Xcat-2. The 3¢UTR is
sufficient for mitochondrial cloud localization.
Note however that the 400 nt luciferase tag is not
as efficiently localized or retained in the cloud as
the smaller transcripts and this is reflected in the
higher background levels. Arrowheads indicate the
mitochondrial cloud. The anti-DIG antibody nonspecifically
reacts with nucleoli (also seen in Fig.
1F). See Fig. 4 for schematic of the constructs
used to generate the injected Xcat-2 transcripts and
for quantitation of localization results. Bar, 80 mm.
Fig. 4. The localization element maps to a 250 nt region within the 3¢UTR. Deletion
mutations used to create mutant transcripts are shown in schematic form. The Xcat-2 ORF
(shaded) and the 3¢UTR (black) of Xcat-2 are shown. Deletions within the 3¢UTR are
indicated by interruptions in the black lines. 50 bp of the 5¢ leader sequence of Xenopus b-
globin cDNA (open box) or 400 bp of the luciferase-coding region (slanted lines) was used
to tag the Xcat-2 3¢UTR. Mitochondrial cloud localization was detected by autoradiography
(for globin-tagged 35S-labeled Xcat-2) or in situ hybridization (for luciferase-tagged Xcat-2)
with DIG-labeled probes. Localization of the encoded transcripts in stage I was scored as
minus as shown in Fig. 3C or D; plus (signal was 2-fold or 4-fold over background) as
represented in Fig. 3A, or plus plus (signal was >5-fold over background) as shown in Fig.
3B. Silver grains over the mitochondrial cloud and an equal area over the nucleus
(background) were counted. Only oocytes with obvious mitochondrial clouds were scored
(n) except for the 5¢ORF construct (*) as the uniform signal made detection of the cloud
difficult in this case. Data are based on two series of injections.
