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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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Hoxb4 overexpression in mouse zygotes delays development.Distribution of 1, 2, 3, and 8-cell embryos at 1.5 or 2.5 days after injection of CMV-IRES-GFP vector (control) or CMV-Hoxb4-IRES-GFP vector (Hoxb4). Notice the high number of 1-cell embryos and the low number of 2-cell embryos at 1.5 days after Hoxb4 injection, compared with control. At 2.5 days after injections, the number of embryos injected with Hoxb4 vector reaching the 8-cell stage is less than 50% of the number obtained after control vector injection. This suggests that there is a delay in early embryo development when Hoxb4 is overexpressed. (*) P value<0.04, as determined by Student's t test.
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pone-0005213-g007: Hoxb4 overexpression in mouse zygotes delays development.Distribution of 1, 2, 3, and 8-cell embryos at 1.5 or 2.5 days after injection of CMV-IRES-GFP vector (control) or CMV-Hoxb4-IRES-GFP vector (Hoxb4). Notice the high number of 1-cell embryos and the low number of 2-cell embryos at 1.5 days after Hoxb4 injection, compared with control. At 2.5 days after injections, the number of embryos injected with Hoxb4 vector reaching the 8-cell stage is less than 50% of the number obtained after control vector injection. This suggests that there is a delay in early embryo development when Hoxb4 is overexpressed. (*) P value<0.04, as determined by Student's t test.

Mentions: In order to test whether the oocyte phenotype of Prep1i/i mice correlates with the increased Hoxb4 mRNA translation, we micro-injected fertilized oocytes from super-ovulated females with either CMV-IRES-GFP or CMV-Hoxb4-IRES-GFP vector and examined their development in culture. The overall death rate due to micro-injection was not significantly different between GFP and Hoxb4 injected zygotes (not shown). Those zygotes lysed within the first 24 hours were not included in the calculations. We performed three series of injections for each vector, using 140 fertilized oocytes with the control and 240 with the Hoxb4 vector. Fluorescence microscopy showed that the GFP was expressed at very low levels in several (although not all) injected zygotes, at the various stages (Fig. S2C). Figure 7 shows the (averaged) results of the three experiments in which at 24 hour intervals the percentage of embryos at each developmental stage (1–2 cells and 3–8 cells) was scored and expressed as percent of the total “live” embryos. Overall, the development was slowed down at all stages in the Hoxb4-microinjected zygotes. The results were statistically significant at the very early (1–2 and 3–8 cells) stages. Overexpression of Hoxb4 showed the same trend also at the morula/blastocyst stage, where it did not reach statistical significance (not shown). We conclude, therefore, that the overexpression of Hoxb4 in mouse zygotes slows down embryo development.


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)

Hoxb4 overexpression in mouse zygotes delays development.Distribution of 1, 2, 3, and 8-cell embryos at 1.5 or 2.5 days after injection of CMV-IRES-GFP vector (control) or CMV-Hoxb4-IRES-GFP vector (Hoxb4). Notice the high number of 1-cell embryos and the low number of 2-cell embryos at 1.5 days after Hoxb4 injection, compared with control. At 2.5 days after injections, the number of embryos injected with Hoxb4 vector reaching the 8-cell stage is less than 50% of the number obtained after control vector injection. This suggests that there is a delay in early embryo development when Hoxb4 is overexpressed. (*) P value<0.04, as determined by Student's t test.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0005213-g007: Hoxb4 overexpression in mouse zygotes delays development.Distribution of 1, 2, 3, and 8-cell embryos at 1.5 or 2.5 days after injection of CMV-IRES-GFP vector (control) or CMV-Hoxb4-IRES-GFP vector (Hoxb4). Notice the high number of 1-cell embryos and the low number of 2-cell embryos at 1.5 days after Hoxb4 injection, compared with control. At 2.5 days after injections, the number of embryos injected with Hoxb4 vector reaching the 8-cell stage is less than 50% of the number obtained after control vector injection. This suggests that there is a delay in early embryo development when Hoxb4 is overexpressed. (*) P value<0.04, as determined by Student's t test.
Mentions: In order to test whether the oocyte phenotype of Prep1i/i mice correlates with the increased Hoxb4 mRNA translation, we micro-injected fertilized oocytes from super-ovulated females with either CMV-IRES-GFP or CMV-Hoxb4-IRES-GFP vector and examined their development in culture. The overall death rate due to micro-injection was not significantly different between GFP and Hoxb4 injected zygotes (not shown). Those zygotes lysed within the first 24 hours were not included in the calculations. We performed three series of injections for each vector, using 140 fertilized oocytes with the control and 240 with the Hoxb4 vector. Fluorescence microscopy showed that the GFP was expressed at very low levels in several (although not all) injected zygotes, at the various stages (Fig. S2C). Figure 7 shows the (averaged) results of the three experiments in which at 24 hour intervals the percentage of embryos at each developmental stage (1–2 cells and 3–8 cells) was scored and expressed as percent of the total “live” embryos. Overall, the development was slowed down at all stages in the Hoxb4-microinjected zygotes. The results were statistically significant at the very early (1–2 and 3–8 cells) stages. Overexpression of Hoxb4 showed the same trend also at the morula/blastocyst stage, where it did not reach statistical significance (not shown). We conclude, therefore, that the overexpression of Hoxb4 in mouse zygotes slows down embryo development.

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