Cytoplasmic Prep1 Interacts with 4EHP Inhibiting Hoxb4 Translation

J Carlos Villaescusa,Francesco Blasi,Dmitry Penkov,Jesús Planagumà,Lisa Mathiasen,Claudia Buratti,Elisabetta Ferretti,Michael Polymenis

doi:10.1371/journal.pone.0005213

Abstract

BackgroundHomeobox 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 FindingsIn 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 Prep1i/i females, accompanied by an increased production of Hoxb4. Finally, Hoxb4 overexpression in mouse zygotes showed a slow in vitro development effect.ConclusionsPrep1 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.

Highlights

Prep1 is a homeodomain transcription factor essential during development [1]
The Prep1-4EHP interaction might bridge the 39UTR of Hoxb4 mRNA to the 59 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
Transcriptional and translational regulation of homeobox genes occurs in Drosophila embryos, where nuclear Bcd regulates the transcription of Hunchback or Even-skipped in the nucleus, while in the cytosol Bcd regulates the translation of Caudal mRNA [8,9,10]

Summary

Introduction

Prep is a homeodomain transcription factor essential during development [1]. A hypomorphic Prep mutation (Prep1i/i) shows variable penetrance and expressivity in mouse, but most Prep1i/i embryos die between E17.5 and P0 [2,3]. Transcriptional and translational regulation of homeobox genes occurs in Drosophila embryos, where nuclear Bcd regulates the transcription of Hunchback or Even-skipped in the nucleus, while in the cytosol Bcd regulates the translation of Caudal (cad) mRNA [8,9,10]. This cytosolic effect is due to the interaction with Drosophila 4EHP (d4EHP) through a YxxxxxxL motif [11] distinct from the consensus binding site for the eukaryotic translation initiation factor 4E (eIF4E), YxxxxLW (where x is any amino acid and W any hydrophobic residue) [12]. Prep contains a putative binding motif for 4EHP, which may reflect a novel unknown function

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