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mRNAs concentrated in specific regions of the oocyte have been found to encode determinants that specify cell fate. We show that an intermediate filament fraction isolated from Xenopus stage VI oocytes specifically contains, in addition to Vg1 RNA, a new localized mRNA, Xcat-2. Like Vg1, Xcat-2 is found in the vegetal cortical region, is inherited by the vegetal blasomeres during development, and is degraded very early in development. Sequence analysis suggests that Xcat-2 encodes a protein that belongs to the CCHC RNA-binding family of zinc finger proteins. Interestingly, the closest known relative to Xcat-2 in this family is nanos, an RNA localized to the posterior pole of the Drosophila oocyte whose protein product suppresses the translation of the transcription factor hunchback. The localized and maternally restricted expression of Xcat-2 RNA suggests a role for its protein in setting up regional differences in gene expression that occur early in development.
Fig. 1. Xcat-2 RNA is highly concentrated in the cytoskeletal
fraction of stage VI oocytes. A northern blot of stage VI total
RNA isolated from the IFF ( ) and SF was probed separately
with Xcat-2, Vg1, and histone H3 32P-labeled DNA. In oocytes,
IFF RNA is four-fold more concentrated in poly(A)+ material as
determined by poly(U) hybridization (data not shown). Therefore,
four-fold more SF RNA (16 mg) was loaded to allow comparison
of mRNA concentration between the SF and IFF. Note that like
Vg1, Xcat-2 (0.9 kb) is highly concentrated in IFF RNA whereas
histone H3 RNA, the internal marker for poly(A)+ material, is of
equal concentration in the two fractions.
Fig. 2. Xcat-2 is localized to the vegetal pole in the stage VI
oocyte and embryo. A northern blot of regional RNA extracted
from cryostat-sectioned oocytes (King and Barklis, 1985) and
embryos was hybridized separately with Xcat-2, Vg1, and histone
H3 32P-labeled DNA. On the left blot, total RNA isolated from
animal pole (An) and vegetal pole (Vg) fifths of stage VI oocytes
is shown. Five-fold less vegetal pole than animal pole poly(A)+
RNA was loaded on the blot (note histone H3 levels), yet Xcat-2
was detected only in the vegetal fifth. On the right blot, each lane
contains total RNA (15 mg) isolated from one fifth of 4- to 8-cell
embryos sectioned along the animal/vegetal axis (An/Vg). Again,
Xcat-2 and Vg1 are found only in the most vegetal fifth (200 mm)
of the embryo.
Fig. 3. In situ hybridization of Xcat-2 in oocyte. Paraffin sections
along the animal-vegetal axis of stage VI oocytes were hybridized
with 35S-labeled Xcat-2 antisense RNA probes. The A/V axis is
indicated by arrowheads with the animal (An) pole at the top and
the vegetal pole (Vg) at the bottom of the figure. The
hybridization signal is represented by silver grains (black) in this
bright-field photograph. The small arrows mark the extent of
cortical hybridization. Autoradiographic exposure was for 14
days.
Fig. 4. Xcat-2 expression is developmentally regulated. (A) RNA
was extracted from the IFF ( ) and SF (unlabeled) of stage VI
oocytes (VI), ovulated eggs (OE), gastrulae (G), and neurulae (N).
The same filters were successively hybridized with 32P-labeled
Vg1, Xcat-2, and H3 histone DNA. Xcat-2 is barely detected by
gastrulation. Note: Autoradiograms were over-exposed to show
any weak responses at later stages of development. (B) RNAase
protection analysis showing Vg1, Xcat-2 and EF1a levels in the
IFF and SF for stage VI oocytes (VI) and ovulated eggs (OE).
Xcat-2 RNA is not released from the IFF at maturation as is Vg1
but remains concentrated in that fraction.
Fig. 5. Diagram and Sequence of the Xcat-2
Transcript. (A) Map of Xcat-2 cDNA (B)
DNA sequence of Xcat-2 with amino acid
single letter translation. The numbering
begins with the 5¢ end of the cDNA clone.
The start and stop codons are in bold type and
the TAAAT polyadenylation signal is
underlined. Potential phosphorylation sites are
circled.
Fig. 6. Comparison between the Xcat-2
and nanos derived protein sequence. (A)
Cartoon of Xcat-2 and nanos protein.
Numbers refer to amino acid position. The
region of homology between the two
proteins is black and the histone-like
region in Xcat-2 is shaded. Putative casein
kinase II sites are indicated by open
rectangles; protein kinase C sites are
denoted by black rectangles. (B) Region of
homology. Identical amino acids are
enclosed in boxes in the two proteins.
Conserved substitutions are marked as a
vertical bar. The black dots mark the
cysteines and histidines within this region
that are hypothesized to form a zinc finger
motif present in both proteins. (C)
Comparison of the Xcat-2 zinc finger motif
with three other zinc finger families: RNAbinding
family (Green and Berg, 1989);
damaged DNA family (Uchida et al., 1987)
and the protein-protein family (Berg,
1990).
Fig. 7. Comparison of a short sequence found in the Xcat-2
protein and selected proteins that interact with nucleic acids. A 10
amino acid stretch in Xcat-2 showed identity with 7 amino acids
(9 allowing conserved substitutions) with a region in histone H1
located within the DNA-binding loop. A search of protein
sequence banks revealed a short list of other proteins 70%
identical with this region; none were completely identical. Many
of these proteins interact with nucleic acids or nucleotides.
Selected examples of such proteins are listed. Amino acids
identical to the Xcat-2 region are boxed, conserved substitutions
are marked with a black dot. The histone H1 sequence is identical
in cow, human, rabbit, chicken, mouse and rat (Allan et al., 1980).
DpnI is a restriction enzyme, endonuclease from Streptococcus
pneumonia. Also listed are AT-BP1, a zinc finger protein found in
rats (Mitchelmore et al., 1991); EF-Tu, an elongation factor
(Jurnak, 1985); LCVLA L protein, a RNA-dependent RNA
polymerase (Singh et al., 1987); NADH dehydrogenase
(ubiquinone).
