XB-ART-35367Differentiation July 1, 2007; 75 (6): 519-28.
Hermes RNA-binding protein targets RNAs-encoding proteins involved in meiotic maturation, early cleavage, and germline development.
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.
PubMed ID: 17309605
Article link: Differentiation
Species referenced: Xenopus
Genes referenced: cat2 mos myc nanos1 rbpms rbpms2
Morpholinos: rbpms2 MO1
Article Images: [+] show captions
|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.|