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Cytoplasmic Prep1 interacts with 4EHP inhibiting Hoxb4 translation.

Villaescusa JC, Buratti C, Penkov D, Mathiasen L, Planagumà J, Ferretti E, Blasi F - PLoS ONE (2009)

Bottom Line: Prep1 has a novel cytoplasmic, 4EHP-dependent, function in the regulation of translation.Mechanistically, the Prep1-4EHP interaction might bridge the 3'UTR of Hoxb4 mRNA to the 5' cap structure.This is the first demonstration that a mammalian homeodomain transcription factor regulates translation, and that this function can be possibly essential for the development of female germ cells and involved in mammalian zygote development.

View Article: PubMed Central - PubMed

Affiliation: IFOM, FIRC Institute of Molecular Oncology, Milano, Italy.

ABSTRACT

Background: Homeobox genes are essential for embryonic patterning and cell fate determination. They are regulated mostly at the transcriptional level. In particular, Prep1 regulates Hox transcription in association with Pbx proteins. Despite its nuclear role as a transcription factor, Prep1 is located in the cytosol of mouse oocytes from primary to antral follicles. The homeodomain factor Bicoid (Bcd) has been shown to interact with 4EHP (eukaryotic translation initiation factor 4E homolog protein) to repress translation of Caudal mRNA and to drive Drosophila embryo development. Interestingly, Prep1 contains a putative binding motif for 4EHP, which may reflect a novel unknown function.

Methodology/principal findings: In this paper we show by confocal microscopy and deconvolution analysis that Prep1 and 4EHP co-localize in the cytosol of growing mouse oocytes, demonstrating their interaction by co-immunoprecipitation and pull-down experiments. A functional 4EHP-binding motif present in Prep1 has been also identified by mutagenesis analysis. Moreover, Prep1 inhibits (>95%) the in vitro translation of a luciferase reporter mRNA fused to the Hoxb4 3'UTR, in the presence of 4EHP. RNA electrophoretic mobility shift assay was used to demonstrate that Prep1 binds the Hoxb4 3'UTR. Furthermore, conventional histology and immunohistochemistry has shown a dramatic oocyte growth failure in hypomorphic mouse Prep1(i/i) females, accompanied by an increased production of Hoxb4. Finally, Hoxb4 overexpression in mouse zygotes showed a slow in vitro development effect.

Conclusions: Prep1 has a novel cytoplasmic, 4EHP-dependent, function in the regulation of translation. Mechanistically, the Prep1-4EHP interaction might bridge the 3'UTR of Hoxb4 mRNA to the 5' cap structure. This is the first demonstration that a mammalian homeodomain transcription factor regulates translation, and that this function can be possibly essential for the development of female germ cells and involved in mammalian zygote development.

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Ovarian phenotype of the Prep1i/i mice.(A) Image showing the developmental failure of Prep1i/i ovaries. Ovaries were smaller in size (compare i/i versus wt), and in almost half of the ovaries analyzed a cyst was observed (arrowhead). (B–D) Haematoxylin and eosin staining of Prep1i/i ovarian sections (C and D) showing the cyst (Cy) formation and the reduced number of follicles compared with Wt (B). Notice that most of the follicles found in Prep1i/i section (D) were primary or secondary follicles. (E) Absence of developed ovary. The asterisk marks the structure that might correspond to the undeveloped ovary. (F) Higher magnification of a non developed ovary, where no follicles were detected. (G–I) Hoxb4 immunostaining of mouse oocytes. Notice the strong staining present in the Prep1i/i oocyte compared with wild-type. The control (I) was performed without primary antibody. Scale bars, B–E 25 µm; F 15 µm; G–I 10 µm.
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pone-0005213-g006: Ovarian phenotype of the Prep1i/i mice.(A) Image showing the developmental failure of Prep1i/i ovaries. Ovaries were smaller in size (compare i/i versus wt), and in almost half of the ovaries analyzed a cyst was observed (arrowhead). (B–D) Haematoxylin and eosin staining of Prep1i/i ovarian sections (C and D) showing the cyst (Cy) formation and the reduced number of follicles compared with Wt (B). Notice that most of the follicles found in Prep1i/i section (D) were primary or secondary follicles. (E) Absence of developed ovary. The asterisk marks the structure that might correspond to the undeveloped ovary. (F) Higher magnification of a non developed ovary, where no follicles were detected. (G–I) Hoxb4 immunostaining of mouse oocytes. Notice the strong staining present in the Prep1i/i oocyte compared with wild-type. The control (I) was performed without primary antibody. Scale bars, B–E 25 µm; F 15 µm; G–I 10 µm.

Mentions: To test for an in vivo role of Prep1 in oocytes and ovary development, we analyzed some of the very few Prep1i/i females that reach adulthood [3]. Because of the low number (n = 5) of available mice, we cannot claim that homozygous Prep1i/i females are sterile, but we have never observed pregnancies in mouse Prep1i/i females. However, Prep1i/i ovaries had a drastic phenotype: they were smaller and underdeveloped (10/10), presented no oocytes (5/10) or developed cysts (4/10) (Fig. 6A–F).


Cytoplasmic Prep1 interacts with 4EHP inhibiting Hoxb4 translation.

Villaescusa JC, Buratti C, Penkov D, Mathiasen L, Planagumà J, Ferretti E, Blasi F - PLoS ONE (2009)

Ovarian phenotype of the Prep1i/i mice.(A) Image showing the developmental failure of Prep1i/i ovaries. Ovaries were smaller in size (compare i/i versus wt), and in almost half of the ovaries analyzed a cyst was observed (arrowhead). (B–D) Haematoxylin and eosin staining of Prep1i/i ovarian sections (C and D) showing the cyst (Cy) formation and the reduced number of follicles compared with Wt (B). Notice that most of the follicles found in Prep1i/i section (D) were primary or secondary follicles. (E) Absence of developed ovary. The asterisk marks the structure that might correspond to the undeveloped ovary. (F) Higher magnification of a non developed ovary, where no follicles were detected. (G–I) Hoxb4 immunostaining of mouse oocytes. Notice the strong staining present in the Prep1i/i oocyte compared with wild-type. The control (I) was performed without primary antibody. Scale bars, B–E 25 µm; F 15 µm; G–I 10 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2664923&req=5

pone-0005213-g006: Ovarian phenotype of the Prep1i/i mice.(A) Image showing the developmental failure of Prep1i/i ovaries. Ovaries were smaller in size (compare i/i versus wt), and in almost half of the ovaries analyzed a cyst was observed (arrowhead). (B–D) Haematoxylin and eosin staining of Prep1i/i ovarian sections (C and D) showing the cyst (Cy) formation and the reduced number of follicles compared with Wt (B). Notice that most of the follicles found in Prep1i/i section (D) were primary or secondary follicles. (E) Absence of developed ovary. The asterisk marks the structure that might correspond to the undeveloped ovary. (F) Higher magnification of a non developed ovary, where no follicles were detected. (G–I) Hoxb4 immunostaining of mouse oocytes. Notice the strong staining present in the Prep1i/i oocyte compared with wild-type. The control (I) was performed without primary antibody. Scale bars, B–E 25 µm; F 15 µm; G–I 10 µm.
Mentions: To test for an in vivo role of Prep1 in oocytes and ovary development, we analyzed some of the very few Prep1i/i females that reach adulthood [3]. Because of the low number (n = 5) of available mice, we cannot claim that homozygous Prep1i/i females are sterile, but we have never observed pregnancies in mouse Prep1i/i females. However, Prep1i/i ovaries had a drastic phenotype: they were smaller and underdeveloped (10/10), presented no oocytes (5/10) or developed cysts (4/10) (Fig. 6A–F).

Bottom Line: Prep1 has a novel cytoplasmic, 4EHP-dependent, function in the regulation of translation.Mechanistically, the Prep1-4EHP interaction might bridge the 3'UTR of Hoxb4 mRNA to the 5' cap structure.This is the first demonstration that a mammalian homeodomain transcription factor regulates translation, and that this function can be possibly essential for the development of female germ cells and involved in mammalian zygote development.

View Article: PubMed Central - PubMed

Affiliation: IFOM, FIRC Institute of Molecular Oncology, Milano, Italy.

ABSTRACT

Background: Homeobox genes are essential for embryonic patterning and cell fate determination. They are regulated mostly at the transcriptional level. In particular, Prep1 regulates Hox transcription in association with Pbx proteins. Despite its nuclear role as a transcription factor, Prep1 is located in the cytosol of mouse oocytes from primary to antral follicles. The homeodomain factor Bicoid (Bcd) has been shown to interact with 4EHP (eukaryotic translation initiation factor 4E homolog protein) to repress translation of Caudal mRNA and to drive Drosophila embryo development. Interestingly, Prep1 contains a putative binding motif for 4EHP, which may reflect a novel unknown function.

Methodology/principal findings: In this paper we show by confocal microscopy and deconvolution analysis that Prep1 and 4EHP co-localize in the cytosol of growing mouse oocytes, demonstrating their interaction by co-immunoprecipitation and pull-down experiments. A functional 4EHP-binding motif present in Prep1 has been also identified by mutagenesis analysis. Moreover, Prep1 inhibits (>95%) the in vitro translation of a luciferase reporter mRNA fused to the Hoxb4 3'UTR, in the presence of 4EHP. RNA electrophoretic mobility shift assay was used to demonstrate that Prep1 binds the Hoxb4 3'UTR. Furthermore, conventional histology and immunohistochemistry has shown a dramatic oocyte growth failure in hypomorphic mouse Prep1(i/i) females, accompanied by an increased production of Hoxb4. Finally, Hoxb4 overexpression in mouse zygotes showed a slow in vitro development effect.

Conclusions: Prep1 has a novel cytoplasmic, 4EHP-dependent, function in the regulation of translation. Mechanistically, the Prep1-4EHP interaction might bridge the 3'UTR of Hoxb4 mRNA to the 5' cap structure. This is the first demonstration that a mammalian homeodomain transcription factor regulates translation, and that this function can be possibly essential for the development of female germ cells and involved in mammalian zygote development.

Show MeSH
Related in: MedlinePlus