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The early development of metazoans is mainly regulated by differential translation and localization of maternal mRNAs in the embryo. In general, these processes are orchestrated by RNA-binding proteins interacting with specific sequence motifs in the 3'-untranslated region (UTR) of their target RNAs. Hermes is an RNA-binding protein, which contains a single RNA recognition motif (RRM) and is found in various vertebrate species from fish to human. In Xenopus laevis, Hermes mRNA and protein are localized in the vegetal region of oocytes. A subpopulation of Hermes protein is concentrated in a specific structure in the vegetal cortex, called the germ plasm (believed to contain determinants of the germ cell fate) where Hermes protein co-localizes with Xcat2 and RINGO/Spy mRNAs. The level of total Hermes protein decreases during maturation. The precocious depletion of Hermes protein by injection of Hermes antisense morpholino oligonucleotide (HE-MO) accelerates the process of maturation and results in cleavage defects in vegetal blastomeres of the embryo. It is known that several maternal mRNAs including RINGO/Spy and Mos are regulated at the translational level during meiotic maturation and early cleavage in Xenopus. The ectopic expression of RINGO/Spy or Mos causes resumption of meiotic maturation and cleavage arrests, which resemble the loss of Hermes phenotypes. We found that the injection of HE-MO enhances the acceleration of maturation caused by the injection of RINGO/Spy mRNA, and that Hermes protein is present as mRNP complex containing RINGO/Spy, Mos, and Xcat2 mRNAs in vivo. We propose that as an RNA-binding protein, Hermes may be involved in maturation, cleavage events at the vegetal pole and germ cell development by negatively regulating the expression of RINGO/Spy, Mos, and Xcat2 mRNAs.
Fig. 1 Temporal expression pattern of Hermes protein. Western
blot analysis of stage VI oocyte, egg, or stage 2–41 embryo extract
using purified Hermes peptide antibody (A), or purified Hermes
protein antibody (B). Note that Hermes protein level decreased
during maturation (between stage VI oocyte and egg). Actin served
as a loading control.
Fig. 3 Cleavage arrests in RINGO/Spy mRNA-injected blastomere.
(A, B) Control embryos in which 5.5 ng of Tumorhead mRNA
was injected into one blastomere at two-cell stage along with
rhodamine-dextran as a lineage tracer. (C, D) Injection of 5.5 ng of
RINGO/Spy mRNA caused cleavage arrest at two-cell or four-cell
stage. The arrows indicate arrested blastomeres and the arrowheads
indicate distribution of dextran tracer within the injected embryos.
Fig. 4 Hermes antisense morpholino oligonucleotide (HE-MO) injection
showed an additive effect on the acceleration of maturation
caused by injection of RINGO/Spy mRNA. (A) Standard control
morpholino (Sc-MO) or HE-MO was injected into 80–100 oocytes
and 48 hr later, 0.5 ng of RINGO/Spy mRNA was injected into
half of the oocytes from each group. Progesterone was added immediately
after RINGO/Spy injection. GVBD was scored at 30 min
intervals (B) Myc-tagged Hermes mRNA rescues the hyperacceleration
of maturation caused by injection of RINGO/Spy mRNA
into HE-MO preinjected oocytes. Oocytes preinjected with Sc-MO,
HE-MO or HE-MO1Myc Hermes mRNA were incubated for
48 hr and then injected with 0.5 ng of RINGO/Spy mRNA.
Fig. 5 Localization of Hermes and RINGO/Spy in the germ plasm
in the mitochondrial cloud. (A) Hermes protein is localized in the
germ plasm within germinal granules (arrows) and in the germ
plasm matrix in stages I–II oocytes (arrowheads). (B) Hermes mRNA
is only present on the germ plasm matrix in stages I–II oocytes
(arrows). (C) Hermes protein is localized in newly forming germinal
granules in prestage I oocytes (arrows). (D) Xcat2 mRNA is present
in germinal granules (arrows) and its distribution resembles that of
Hermes protein in stages I–II oocytes (compared to (A)). (E)
RINGO/Spy mRNA is present in germ plasm outside of germinal
granules in stages I–II oocytes (arrows). Scale bars are equal to
500 nm. Electron microscopy immunostaining was performed using
Hermes rabbit polyclonal antibody, anti-rabbit nanogold conjugated
secondary antibody and silver enhanced. Electron microscopy
in situ hybridization was performed with antisense digoxigenin
labeled RNA probes of Hermes, Xcat2 or RINGO/Spy and visualized
using the nanogold conjugated anti-digoxigenin antibody
and silver enhancement. (F–H) Localization of RINGO/Spy in oocytes.
(F) In prestage I oocytes, a large population of RINGO/Spy
mRNA is located in the mitochondrial cloud (arrows). (G) In late
stage II/stage III oocytes, RINGO/Spy mRNA moves toward the
vegetal pole (arrow) and is also present in the cytoplasm and vegetal
cortex (arrowheads). (H) In stage II oocytes, RINGO/Spy
mRNA is localized as a disc at the vegetal pole region (arrows).
In situ hybridization, whole-mount (F, H) or on sections (G) was
performed on albino oocytes.
Fig. 6 Hermes protein binds to RINGO/Spy, Mos, and Xcat2
mRNAs in vivo. Noninjected control oocytes and oocytes injected
with mRNAs encoding myc-tagged Hermes were cultured overnight.
RNPs were immunoprecipitated from oocyte lysates by Myc
antibody (Myc IP) or nonspecific immunoglobulin G (IgG IP).
RNA was isolated from the immunoprecipitates and subjected to
RT-PCR using specific primers for RINGO/Spy, Mos, Xcat2,
eIF4E, and Vg1 as described in ‘‘Materials and methods.’’ Also
shown are the RT-PCR reactions for the total RNAs isolated from
oocyte lysate.
Fig. 7 Summary and model of predicted
Hermes functions. (A) In oocytes, Hermes
protein may repress translation of Xcat2,
RINGO/Spy, and Mos mRNAs which are
involved in maturation, early cleavage and
germ cell development. (B) Hermes multimers
and cofactor complexes bind to the
30 UTR of targeted mRNAs. The Hermes
complex cooperates with other translation
repressors and blocks access of translation
initiation factors. (C) Hermes triggers formation
of multimeric RNP particles, which are
inaccessible to the translational machinery.
ORF, open reading frame; HE, Hermes;
Co-HE, Hermes cofactor.
