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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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Hoxb cluster expression in oocytes and associated granulosa cells (OGC) and Hoxb mRNA immunoprecipitation by anti-Prep1 antibodies.(A) Expression of Hoxb genes in OGC cells by RT-PCR analysis. Notice that 5 different Hoxb genes (Hoxb4–8) are expressed. (B) Extracts from crosslinked ovarian cells (see Materials and Methods) were immunoprecipitated with anti-Prep1 or not related (NR) antibodies. The RNA was extracted and subjected to RT-PCR with degenerated Antennapedia primers (upper part), which amplified Hox messengers (HoxA and B clusters). After cloning and sequencing of the amplicons, Hoxb4 was highly represented among the amplicons. Then, specific Hoxb4 primers were used to confirm the previous result (middle part), amplifying Hoxb4 mRNA from the OGC co-immunoprecipitated RNA. Notice that specific primers for Hoxb5, which is expressed in OGC but was not identified among the Hox amplicons, is not amplified from the OGC co-immunoprecipitated RNA (lower part).
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pone-0005213-g004: Hoxb cluster expression in oocytes and associated granulosa cells (OGC) and Hoxb mRNA immunoprecipitation by anti-Prep1 antibodies.(A) Expression of Hoxb genes in OGC cells by RT-PCR analysis. Notice that 5 different Hoxb genes (Hoxb4–8) are expressed. (B) Extracts from crosslinked ovarian cells (see Materials and Methods) were immunoprecipitated with anti-Prep1 or not related (NR) antibodies. The RNA was extracted and subjected to RT-PCR with degenerated Antennapedia primers (upper part), which amplified Hox messengers (HoxA and B clusters). After cloning and sequencing of the amplicons, Hoxb4 was highly represented among the amplicons. Then, specific Hoxb4 primers were used to confirm the previous result (middle part), amplifying Hoxb4 mRNA from the OGC co-immunoprecipitated RNA. Notice that specific primers for Hoxb5, which is expressed in OGC but was not identified among the Hox amplicons, is not amplified from the OGC co-immunoprecipitated RNA (lower part).

Mentions: RT-PCR analysis with specific primers shows that Hoxb4, 5, 6, 7 and 8 are expressed in the oocyte and associated ganulosa cells (OGC, Fig. 4A). To test whether Prep1 binds mRNAs coding for Hox genes, we immunoprecipitated crosslinked RNA from OGC using a Prep1 antibody (see Material and methods). Degenerated primers (HoxA and HoxB) based on an early nucleotide consensus for vertebrate Antennapedia class homeodomains [18], [22], [23] (see Materials section) were used to amplify homeobox sequences in the co-immunoprecipitated RNA from OGC. As shown in the top line of Fig. 4B, Hox amplicons were detected by PCR, meaning that Hox RNAs were co-immunoprecipitated by Prep1. After cloning and sequencing those amplicons, we found Hoxb4 and Hoxb8 sequences highly represented among the different clones. Knowing that Hoxb4 and Hoxb8 mRNAs can be co-immunoprecipitated by Prep1, we used specific primers to confirm this result. In fact, we were able to amplify Hoxb4 and Hoxb8 from the co-immunoprecipitated OGC RNA (Fig. 4B, second line, and data not shown for Hoxb8). In contrast, we could not amplify other Hox members from the co-immunoprecipitated RNA, such as Hoxb5 (third line, Fig. 4B), which was present in OGC extracts (Fig. 4A). Prep1, therefore, associates at least to Hoxb4 and Hoxb8 mRNA in oocyte- associated granulosa cells.


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)

Hoxb cluster expression in oocytes and associated granulosa cells (OGC) and Hoxb mRNA immunoprecipitation by anti-Prep1 antibodies.(A) Expression of Hoxb genes in OGC cells by RT-PCR analysis. Notice that 5 different Hoxb genes (Hoxb4–8) are expressed. (B) Extracts from crosslinked ovarian cells (see Materials and Methods) were immunoprecipitated with anti-Prep1 or not related (NR) antibodies. The RNA was extracted and subjected to RT-PCR with degenerated Antennapedia primers (upper part), which amplified Hox messengers (HoxA and B clusters). After cloning and sequencing of the amplicons, Hoxb4 was highly represented among the amplicons. Then, specific Hoxb4 primers were used to confirm the previous result (middle part), amplifying Hoxb4 mRNA from the OGC co-immunoprecipitated RNA. Notice that specific primers for Hoxb5, which is expressed in OGC but was not identified among the Hox amplicons, is not amplified from the OGC co-immunoprecipitated RNA (lower part).
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pone-0005213-g004: Hoxb cluster expression in oocytes and associated granulosa cells (OGC) and Hoxb mRNA immunoprecipitation by anti-Prep1 antibodies.(A) Expression of Hoxb genes in OGC cells by RT-PCR analysis. Notice that 5 different Hoxb genes (Hoxb4–8) are expressed. (B) Extracts from crosslinked ovarian cells (see Materials and Methods) were immunoprecipitated with anti-Prep1 or not related (NR) antibodies. The RNA was extracted and subjected to RT-PCR with degenerated Antennapedia primers (upper part), which amplified Hox messengers (HoxA and B clusters). After cloning and sequencing of the amplicons, Hoxb4 was highly represented among the amplicons. Then, specific Hoxb4 primers were used to confirm the previous result (middle part), amplifying Hoxb4 mRNA from the OGC co-immunoprecipitated RNA. Notice that specific primers for Hoxb5, which is expressed in OGC but was not identified among the Hox amplicons, is not amplified from the OGC co-immunoprecipitated RNA (lower part).
Mentions: RT-PCR analysis with specific primers shows that Hoxb4, 5, 6, 7 and 8 are expressed in the oocyte and associated ganulosa cells (OGC, Fig. 4A). To test whether Prep1 binds mRNAs coding for Hox genes, we immunoprecipitated crosslinked RNA from OGC using a Prep1 antibody (see Material and methods). Degenerated primers (HoxA and HoxB) based on an early nucleotide consensus for vertebrate Antennapedia class homeodomains [18], [22], [23] (see Materials section) were used to amplify homeobox sequences in the co-immunoprecipitated RNA from OGC. As shown in the top line of Fig. 4B, Hox amplicons were detected by PCR, meaning that Hox RNAs were co-immunoprecipitated by Prep1. After cloning and sequencing those amplicons, we found Hoxb4 and Hoxb8 sequences highly represented among the different clones. Knowing that Hoxb4 and Hoxb8 mRNAs can be co-immunoprecipitated by Prep1, we used specific primers to confirm this result. In fact, we were able to amplify Hoxb4 and Hoxb8 from the co-immunoprecipitated OGC RNA (Fig. 4B, second line, and data not shown for Hoxb8). In contrast, we could not amplify other Hox members from the co-immunoprecipitated RNA, such as Hoxb5 (third line, Fig. 4B), which was present in OGC extracts (Fig. 4A). Prep1, therefore, associates at least to Hoxb4 and Hoxb8 mRNA in oocyte- associated granulosa cells.

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