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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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Prep1 inhibits translation of the luciferase-Hoxb4 mRNA and is able to bind the 3′-UTR region of Hoxb4 mRNA.(A) Luc-Hoxb4 3′UTR mRNA was translated in vitro in a rabbit reticulocytes system with the additions indicated at the bottom (Prep1 or 4EHP previously in vitro synthesized under T7 promoter). Luciferase activity is expressed in percent of the control; the 100% value is the level of luciferase translated in the absence of any added protein (column 6). Addition of in vitro translated Prep1 inhibits Luc-Hoxb4 3′UTR mRNA translation (columns 1 and 4) while no further effect is observed when the in vitro translated 4EHP protein is added to the reaction with Prep1 (column 1) or alone (column 3). Less inhibition is obtained with the in vitro translated Prep1 4EHP-binding (Y-LL) mutant (Prep1-Mut) (columns 2 and 5). N-values are 5. (B) This control shows that the differences observed in (A) are not due to interference with the in vitro transcription of the luciferase-Hoxb4 3′UTR mRNA. RT-PCR analysis of Luc-Hoxb4 3′UTR mRNA present in samples 1–6 (A). Each reaction was amplified for 25 and 30 cycles. Notice the absence of amplification in the RT(-), indicating that the plasmid used for Luc-Hoxb4 3′UTR transcription had been completely digested by the DNAse treatment. (C) Prep1 does not inhibit translation of Luc-Cdx2 3′UTR mRNA, independently of the presence of 4EHP. Thus the inhibitory effect appears to be dictated by the presence of the Hoxb4 3′UTR. N-values are 3. (D) Prep1 inhibits in vitro translation of Luc-Hoxb4 3′UTR mRNA in a dose-dependent manner. Compare non diluted Prep1 (column 1) with dilutions 1/2 and 1/5 (columns 2 and 3). N-values are 4. (E) Anti-4EHP antibodies prevent the inhibitory action of Prep1. Inhibition of Luc-Hoxb4 3′UTR mRNA translation by Prep1 was reversed when 2 µg of anti-4EHP (but not an unrelated) antibody was added to the reaction. N-values are 3. (F) RNA-EMSA showing specific Prep1 binding to Hoxb4 3′UTR. First lane shows the [alpha-32P] rUTP-labelled probe. Second lane shows the shift induced by the addition of Prep1 to the reaction (arrow). Third lane shows the induction of a super-shift by the Prep1 antibody (arrowhead). Lane 4 shows that the effect of the antibody is specific since an anti-Pbx antibody has no effect. Same experiment using antisense probe is shown in lanes 5–8.
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pone-0005213-g005: Prep1 inhibits translation of the luciferase-Hoxb4 mRNA and is able to bind the 3′-UTR region of Hoxb4 mRNA.(A) Luc-Hoxb4 3′UTR mRNA was translated in vitro in a rabbit reticulocytes system with the additions indicated at the bottom (Prep1 or 4EHP previously in vitro synthesized under T7 promoter). Luciferase activity is expressed in percent of the control; the 100% value is the level of luciferase translated in the absence of any added protein (column 6). Addition of in vitro translated Prep1 inhibits Luc-Hoxb4 3′UTR mRNA translation (columns 1 and 4) while no further effect is observed when the in vitro translated 4EHP protein is added to the reaction with Prep1 (column 1) or alone (column 3). Less inhibition is obtained with the in vitro translated Prep1 4EHP-binding (Y-LL) mutant (Prep1-Mut) (columns 2 and 5). N-values are 5. (B) This control shows that the differences observed in (A) are not due to interference with the in vitro transcription of the luciferase-Hoxb4 3′UTR mRNA. RT-PCR analysis of Luc-Hoxb4 3′UTR mRNA present in samples 1–6 (A). Each reaction was amplified for 25 and 30 cycles. Notice the absence of amplification in the RT(-), indicating that the plasmid used for Luc-Hoxb4 3′UTR transcription had been completely digested by the DNAse treatment. (C) Prep1 does not inhibit translation of Luc-Cdx2 3′UTR mRNA, independently of the presence of 4EHP. Thus the inhibitory effect appears to be dictated by the presence of the Hoxb4 3′UTR. N-values are 3. (D) Prep1 inhibits in vitro translation of Luc-Hoxb4 3′UTR mRNA in a dose-dependent manner. Compare non diluted Prep1 (column 1) with dilutions 1/2 and 1/5 (columns 2 and 3). N-values are 4. (E) Anti-4EHP antibodies prevent the inhibitory action of Prep1. Inhibition of Luc-Hoxb4 3′UTR mRNA translation by Prep1 was reversed when 2 µg of anti-4EHP (but not an unrelated) antibody was added to the reaction. N-values are 3. (F) RNA-EMSA showing specific Prep1 binding to Hoxb4 3′UTR. First lane shows the [alpha-32P] rUTP-labelled probe. Second lane shows the shift induced by the addition of Prep1 to the reaction (arrow). Third lane shows the induction of a super-shift by the Prep1 antibody (arrowhead). Lane 4 shows that the effect of the antibody is specific since an anti-Pbx antibody has no effect. Same experiment using antisense probe is shown in lanes 5–8.

Mentions: Since Prep1 can bind both some mRNAs and the 4EHP translation inhibitor, we studied the effect of the Prep1-4EHP complex on Hoxb4 mRNA translation in vitro using a rabbit reticulocytes lysate translation system. We cloned the 3′UTR of Hoxb4 at the 3′ end of a luciferase reporter gene, expressed under the SP6 promoter (Luc-3′Hoxb4). As shown in Fig. 5A (n-values = 5), addition of in vitro-translated Prep1 (previously synthesized under the T7 promoter) inhibited Luc-3′Hoxb4 translation by more than 90% (column 1 versus 6). In contrast, the Prep1 mutant (Prep1 YLL) inhibited only around 40% (column 2). This result completely agrees with the capacity of Prep1-YLL to bind 4EHP, which is low but not completely abolished (Fig. 3C). Addition of exogenous 4EHP to the reaction apparently had no major effect on Luc-3′Hoxb4 mRNA translation (see columns 1, 4, Fig. 5A). However, we suspected that 4EHP may already be present in excess in the rabbit reticulocyte lysate. In fact, western blot analysis identified 4EHP in rabbit reticulocyte lysates (data not shown). Moreover, RT-PCR identified 4EHP mRNA in the micrococcal nuclease-untreated rabbit reticulocyte lysate (Fig. S1E) . To verify that the inhibitory effect of Prep1 was not due to a difference in the amount of RNA produced in the reaction, we also extracted total RNA from the samples shown in Fig. 5A and analysed the amount of Luc-3′Hoxb4 mRNA by RT-PCR (at 25 and 30 cycles). The amount of Luc-3′Hoxb4 mRNA produced in each reaction was comparable in all cases in non saturated PCR cycles (Fig. 5B), suggesting that the strong differences observed in Fig. 5A cannot be explained by a differential RNA production between reactions. Moreover, the amount of Prep1 or Prep1-YLL protein added to the reactions was comparable (Fig. S1D). We also verified that the inhibitory effect of Prep1 was specific for Hoxb4 3′-UTR. Indeed, translation of a Luc-3′Cdx2 mRNA, containing the 3′UTR of the mammalian ortholog of Caudal Cdx2 [26], was only marginally affected by Prep1 (Fig. 5C, n = 3). Finally, we also show that the inhibition of Luc-3′Hoxb4 mRNA by Prep1 is dose-dependent (compare columns 1, 2 and 3 with column 4 on Fig. 5D, n = 4).


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)

Prep1 inhibits translation of the luciferase-Hoxb4 mRNA and is able to bind the 3′-UTR region of Hoxb4 mRNA.(A) Luc-Hoxb4 3′UTR mRNA was translated in vitro in a rabbit reticulocytes system with the additions indicated at the bottom (Prep1 or 4EHP previously in vitro synthesized under T7 promoter). Luciferase activity is expressed in percent of the control; the 100% value is the level of luciferase translated in the absence of any added protein (column 6). Addition of in vitro translated Prep1 inhibits Luc-Hoxb4 3′UTR mRNA translation (columns 1 and 4) while no further effect is observed when the in vitro translated 4EHP protein is added to the reaction with Prep1 (column 1) or alone (column 3). Less inhibition is obtained with the in vitro translated Prep1 4EHP-binding (Y-LL) mutant (Prep1-Mut) (columns 2 and 5). N-values are 5. (B) This control shows that the differences observed in (A) are not due to interference with the in vitro transcription of the luciferase-Hoxb4 3′UTR mRNA. RT-PCR analysis of Luc-Hoxb4 3′UTR mRNA present in samples 1–6 (A). Each reaction was amplified for 25 and 30 cycles. Notice the absence of amplification in the RT(-), indicating that the plasmid used for Luc-Hoxb4 3′UTR transcription had been completely digested by the DNAse treatment. (C) Prep1 does not inhibit translation of Luc-Cdx2 3′UTR mRNA, independently of the presence of 4EHP. Thus the inhibitory effect appears to be dictated by the presence of the Hoxb4 3′UTR. N-values are 3. (D) Prep1 inhibits in vitro translation of Luc-Hoxb4 3′UTR mRNA in a dose-dependent manner. Compare non diluted Prep1 (column 1) with dilutions 1/2 and 1/5 (columns 2 and 3). N-values are 4. (E) Anti-4EHP antibodies prevent the inhibitory action of Prep1. Inhibition of Luc-Hoxb4 3′UTR mRNA translation by Prep1 was reversed when 2 µg of anti-4EHP (but not an unrelated) antibody was added to the reaction. N-values are 3. (F) RNA-EMSA showing specific Prep1 binding to Hoxb4 3′UTR. First lane shows the [alpha-32P] rUTP-labelled probe. Second lane shows the shift induced by the addition of Prep1 to the reaction (arrow). Third lane shows the induction of a super-shift by the Prep1 antibody (arrowhead). Lane 4 shows that the effect of the antibody is specific since an anti-Pbx antibody has no effect. Same experiment using antisense probe is shown in lanes 5–8.
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Related In: Results  -  Collection

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

pone-0005213-g005: Prep1 inhibits translation of the luciferase-Hoxb4 mRNA and is able to bind the 3′-UTR region of Hoxb4 mRNA.(A) Luc-Hoxb4 3′UTR mRNA was translated in vitro in a rabbit reticulocytes system with the additions indicated at the bottom (Prep1 or 4EHP previously in vitro synthesized under T7 promoter). Luciferase activity is expressed in percent of the control; the 100% value is the level of luciferase translated in the absence of any added protein (column 6). Addition of in vitro translated Prep1 inhibits Luc-Hoxb4 3′UTR mRNA translation (columns 1 and 4) while no further effect is observed when the in vitro translated 4EHP protein is added to the reaction with Prep1 (column 1) or alone (column 3). Less inhibition is obtained with the in vitro translated Prep1 4EHP-binding (Y-LL) mutant (Prep1-Mut) (columns 2 and 5). N-values are 5. (B) This control shows that the differences observed in (A) are not due to interference with the in vitro transcription of the luciferase-Hoxb4 3′UTR mRNA. RT-PCR analysis of Luc-Hoxb4 3′UTR mRNA present in samples 1–6 (A). Each reaction was amplified for 25 and 30 cycles. Notice the absence of amplification in the RT(-), indicating that the plasmid used for Luc-Hoxb4 3′UTR transcription had been completely digested by the DNAse treatment. (C) Prep1 does not inhibit translation of Luc-Cdx2 3′UTR mRNA, independently of the presence of 4EHP. Thus the inhibitory effect appears to be dictated by the presence of the Hoxb4 3′UTR. N-values are 3. (D) Prep1 inhibits in vitro translation of Luc-Hoxb4 3′UTR mRNA in a dose-dependent manner. Compare non diluted Prep1 (column 1) with dilutions 1/2 and 1/5 (columns 2 and 3). N-values are 4. (E) Anti-4EHP antibodies prevent the inhibitory action of Prep1. Inhibition of Luc-Hoxb4 3′UTR mRNA translation by Prep1 was reversed when 2 µg of anti-4EHP (but not an unrelated) antibody was added to the reaction. N-values are 3. (F) RNA-EMSA showing specific Prep1 binding to Hoxb4 3′UTR. First lane shows the [alpha-32P] rUTP-labelled probe. Second lane shows the shift induced by the addition of Prep1 to the reaction (arrow). Third lane shows the induction of a super-shift by the Prep1 antibody (arrowhead). Lane 4 shows that the effect of the antibody is specific since an anti-Pbx antibody has no effect. Same experiment using antisense probe is shown in lanes 5–8.
Mentions: Since Prep1 can bind both some mRNAs and the 4EHP translation inhibitor, we studied the effect of the Prep1-4EHP complex on Hoxb4 mRNA translation in vitro using a rabbit reticulocytes lysate translation system. We cloned the 3′UTR of Hoxb4 at the 3′ end of a luciferase reporter gene, expressed under the SP6 promoter (Luc-3′Hoxb4). As shown in Fig. 5A (n-values = 5), addition of in vitro-translated Prep1 (previously synthesized under the T7 promoter) inhibited Luc-3′Hoxb4 translation by more than 90% (column 1 versus 6). In contrast, the Prep1 mutant (Prep1 YLL) inhibited only around 40% (column 2). This result completely agrees with the capacity of Prep1-YLL to bind 4EHP, which is low but not completely abolished (Fig. 3C). Addition of exogenous 4EHP to the reaction apparently had no major effect on Luc-3′Hoxb4 mRNA translation (see columns 1, 4, Fig. 5A). However, we suspected that 4EHP may already be present in excess in the rabbit reticulocyte lysate. In fact, western blot analysis identified 4EHP in rabbit reticulocyte lysates (data not shown). Moreover, RT-PCR identified 4EHP mRNA in the micrococcal nuclease-untreated rabbit reticulocyte lysate (Fig. S1E) . To verify that the inhibitory effect of Prep1 was not due to a difference in the amount of RNA produced in the reaction, we also extracted total RNA from the samples shown in Fig. 5A and analysed the amount of Luc-3′Hoxb4 mRNA by RT-PCR (at 25 and 30 cycles). The amount of Luc-3′Hoxb4 mRNA produced in each reaction was comparable in all cases in non saturated PCR cycles (Fig. 5B), suggesting that the strong differences observed in Fig. 5A cannot be explained by a differential RNA production between reactions. Moreover, the amount of Prep1 or Prep1-YLL protein added to the reactions was comparable (Fig. S1D). We also verified that the inhibitory effect of Prep1 was specific for Hoxb4 3′-UTR. Indeed, translation of a Luc-3′Cdx2 mRNA, containing the 3′UTR of the mammalian ortholog of Caudal Cdx2 [26], was only marginally affected by Prep1 (Fig. 5C, n = 3). Finally, we also show that the inhibition of Luc-3′Hoxb4 mRNA by Prep1 is dose-dependent (compare columns 1, 2 and 3 with column 4 on Fig. 5D, n = 4).

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