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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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Immunofluorescence and deconvolution analysis of Prep1 and 4EHP expression in mouse ovarian follicles.(A–D) A primary follicle. The cuboidal shape of the sourrounding granulosa cells (gc) indicates the activation of the follicle. 4EHP and Prep1 are both located in the cytosol of the oocyte (oo) and colocalize (Merge, C). (E–H) Secondary (arrow) and antral (asterisk) follicles show Prep1 expression in the nucleus of granulosa cells. In contrast, 4EHP is always cytosolic and no co-localization is evident (panel G, Merge). (I–L) Granulosa cells from an antral follicle showing cytosolic localization of 4EHP. In contrast, Prep1 was clearly localized into the nucleus of the cells. Notice the absence of co-localization in the cytosol (Merge, K). (M–P) Deconvolution analysis of Prep1-4EHP localization in the cytosol of an antral oocyte. 3D co-localization analyses of 4EHP and Prep1 were performed on a voxel-to-voxel basis using automatic threshold co-localization algorithm by Costes and Locket. The image stacks obtained by confocal microscopy were deconvolved with 20 iterations using theoretical point spread function and maximum likelihood estimation algorithms of Huygens Essential software (see Materials and Methods). Notice the co-localization in white (O–P). Sale bars, D 10 µm; H 25 µm; L 15 µm; P 5 µm.
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pone-0005213-g002: Immunofluorescence and deconvolution analysis of Prep1 and 4EHP expression in mouse ovarian follicles.(A–D) A primary follicle. The cuboidal shape of the sourrounding granulosa cells (gc) indicates the activation of the follicle. 4EHP and Prep1 are both located in the cytosol of the oocyte (oo) and colocalize (Merge, C). (E–H) Secondary (arrow) and antral (asterisk) follicles show Prep1 expression in the nucleus of granulosa cells. In contrast, 4EHP is always cytosolic and no co-localization is evident (panel G, Merge). (I–L) Granulosa cells from an antral follicle showing cytosolic localization of 4EHP. In contrast, Prep1 was clearly localized into the nucleus of the cells. Notice the absence of co-localization in the cytosol (Merge, K). (M–P) Deconvolution analysis of Prep1-4EHP localization in the cytosol of an antral oocyte. 3D co-localization analyses of 4EHP and Prep1 were performed on a voxel-to-voxel basis using automatic threshold co-localization algorithm by Costes and Locket. The image stacks obtained by confocal microscopy were deconvolved with 20 iterations using theoretical point spread function and maximum likelihood estimation algorithms of Huygens Essential software (see Materials and Methods). Notice the co-localization in white (O–P). Sale bars, D 10 µm; H 25 µm; L 15 µm; P 5 µm.

Mentions: Prep1 and 4EHP co-localize in the cytoplasm of mouse primary oocytes (oo), as shown by confocal immunofluorescence analysis (Fig. 2A–D). From secondary to antral follicles, Prep1 is located in the nucleus of granulosa cells (gc), where 4EHP is mainly cytosolic, and no co-localization is observed (Fig. 2E–L). In contrast, Prep1 and 4EHP still co-localize in the cytosol of oocytes from secondary to antral follicles. The co-localization between Prep1 and 4EHP in the cytosol of antral oocytes is confirmed (Fig. 2M–P) by deconvolution analysis, which increases image resolution and decreases false positives [19]. As it is shown in Fig. S1A, the 4EHP antibody specifically detects 4EHP but not its close homolog eIF4E. In the case of Prep1 antibody, its specificity has been described previously [2], [3].


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)

Immunofluorescence and deconvolution analysis of Prep1 and 4EHP expression in mouse ovarian follicles.(A–D) A primary follicle. The cuboidal shape of the sourrounding granulosa cells (gc) indicates the activation of the follicle. 4EHP and Prep1 are both located in the cytosol of the oocyte (oo) and colocalize (Merge, C). (E–H) Secondary (arrow) and antral (asterisk) follicles show Prep1 expression in the nucleus of granulosa cells. In contrast, 4EHP is always cytosolic and no co-localization is evident (panel G, Merge). (I–L) Granulosa cells from an antral follicle showing cytosolic localization of 4EHP. In contrast, Prep1 was clearly localized into the nucleus of the cells. Notice the absence of co-localization in the cytosol (Merge, K). (M–P) Deconvolution analysis of Prep1-4EHP localization in the cytosol of an antral oocyte. 3D co-localization analyses of 4EHP and Prep1 were performed on a voxel-to-voxel basis using automatic threshold co-localization algorithm by Costes and Locket. The image stacks obtained by confocal microscopy were deconvolved with 20 iterations using theoretical point spread function and maximum likelihood estimation algorithms of Huygens Essential software (see Materials and Methods). Notice the co-localization in white (O–P). Sale bars, D 10 µm; H 25 µm; L 15 µm; P 5 µm.
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Related In: Results  -  Collection

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pone-0005213-g002: Immunofluorescence and deconvolution analysis of Prep1 and 4EHP expression in mouse ovarian follicles.(A–D) A primary follicle. The cuboidal shape of the sourrounding granulosa cells (gc) indicates the activation of the follicle. 4EHP and Prep1 are both located in the cytosol of the oocyte (oo) and colocalize (Merge, C). (E–H) Secondary (arrow) and antral (asterisk) follicles show Prep1 expression in the nucleus of granulosa cells. In contrast, 4EHP is always cytosolic and no co-localization is evident (panel G, Merge). (I–L) Granulosa cells from an antral follicle showing cytosolic localization of 4EHP. In contrast, Prep1 was clearly localized into the nucleus of the cells. Notice the absence of co-localization in the cytosol (Merge, K). (M–P) Deconvolution analysis of Prep1-4EHP localization in the cytosol of an antral oocyte. 3D co-localization analyses of 4EHP and Prep1 were performed on a voxel-to-voxel basis using automatic threshold co-localization algorithm by Costes and Locket. The image stacks obtained by confocal microscopy were deconvolved with 20 iterations using theoretical point spread function and maximum likelihood estimation algorithms of Huygens Essential software (see Materials and Methods). Notice the co-localization in white (O–P). Sale bars, D 10 µm; H 25 µm; L 15 µm; P 5 µm.
Mentions: Prep1 and 4EHP co-localize in the cytoplasm of mouse primary oocytes (oo), as shown by confocal immunofluorescence analysis (Fig. 2A–D). From secondary to antral follicles, Prep1 is located in the nucleus of granulosa cells (gc), where 4EHP is mainly cytosolic, and no co-localization is observed (Fig. 2E–L). In contrast, Prep1 and 4EHP still co-localize in the cytosol of oocytes from secondary to antral follicles. The co-localization between Prep1 and 4EHP in the cytosol of antral oocytes is confirmed (Fig. 2M–P) by deconvolution analysis, which increases image resolution and decreases false positives [19]. As it is shown in Fig. S1A, the 4EHP antibody specifically detects 4EHP but not its close homolog eIF4E. In the case of Prep1 antibody, its specificity has been described previously [2], [3].

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