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Differential RNA-binding activity of the hnRNP G protein correlated with the sex genotype in the amphibian oocyte.

Kanhoush R, Praseuth D, Perrin C, Chardard D, Vinh J, Penrad-Mobayed M - Nucleic Acids Res. (2011)

Bottom Line: A proteomic approach has enabled the identification of an orthologue of the splicing factor hnRNP G in the amphibians Xenopus tropicalis, Ambystoma mexicanum, Notophthalmus viridescens and Pleurodeles walt, which shows a specific RNA-binding affinity similar to that of the human hnRN G protein.In situ hybridization to lampbrush chromosomes of P. waltl revealed the presence of a family of hnRNP G genes, which were mapped on the Z and W chromosomes and one autosome.This indicates that the isoforms identified in this study are possibly encoded by a gene family linked to the evolution of sex chromosomes similarly to the hnRNP G/RBMX gene family in mammals.

View Article: PubMed Central - PubMed

Affiliation: Institut Jacques Monod, UMR 7592, CNRS and Université Paris-Diderot, Museum National d'Histoire Naturelle, U 565, USM 503, UMR 5153, INSERM and CNRS, Paris, France.

ABSTRACT
A proteomic approach has enabled the identification of an orthologue of the splicing factor hnRNP G in the amphibians Xenopus tropicalis, Ambystoma mexicanum, Notophthalmus viridescens and Pleurodeles walt, which shows a specific RNA-binding affinity similar to that of the human hnRN G protein. Three isoforms of this protein with a differential binding affinity for a specific RNA probe were identified in the P. walt oocyte. In situ hybridization to lampbrush chromosomes of P. waltl revealed the presence of a family of hnRNP G genes, which were mapped on the Z and W chromosomes and one autosome. This indicates that the isoforms identified in this study are possibly encoded by a gene family linked to the evolution of sex chromosomes similarly to the hnRNP G/RBMX gene family in mammals.

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Interaction of the P. waltl hnRNP G isoforms with the WEc RNA. (A) 2D-NWA of the 32P-labelled RNA WEc probe with nuclear proteins from ZZ, ZW and WW GVs, followed by immunodetection using the anti-hnRNP G serum. Note that the homologues of hnRNPG formed a train of basic spots ranging from pI 9 to 10 with two major spots at pI 10. The most basic spot corresponded to the one detected by NWA in ZW and WW GVs. (B) An example of isoelectrofocusing of the protein extracts of the ZW GVs showing three spots of hnRNP G at pI 10 (arrowheads) in the silver stained gel (S). Note that only one spot was visible in the northwestern blot (N).
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Figure 5: Interaction of the P. waltl hnRNP G isoforms with the WEc RNA. (A) 2D-NWA of the 32P-labelled RNA WEc probe with nuclear proteins from ZZ, ZW and WW GVs, followed by immunodetection using the anti-hnRNP G serum. Note that the homologues of hnRNPG formed a train of basic spots ranging from pI 9 to 10 with two major spots at pI 10. The most basic spot corresponded to the one detected by NWA in ZW and WW GVs. (B) An example of isoelectrofocusing of the protein extracts of the ZW GVs showing three spots of hnRNP G at pI 10 (arrowheads) in the silver stained gel (S). Note that only one spot was visible in the northwestern blot (N).

Mentions: Northwestern and immunodetection assays performed in parallel on the same 2D western blot further confirmed the identification of the 42 kDa/pI 10 P. waltl polypeptides as hnRNPG homologues (Figure 5A). The position of the immunodetected spots corresponded to the polypeptides showed by mass spectrometry to be the homologues of the human hnRNPG protein. Furthermore, the western blot analysis revealed the existence in the oocytes of the three karyotypes, of a basic train of isoforms, ranging from pI 9 to 10 with closely similar molecular weights and present in different amounts. Three major spots were visible, one at pI 9 and the other two at pI 10, which could be distinguished from one another by a slight variation in molecular weight and pI. The superimposition of the autoradiogram with the western blot showed that none of the hnRNP G homologues bound the WEc RNA probe in the ZZ GVs because no radioactive hybridization signals were detected in the corresponding area of the autoradiogram. In contrast, the most basic hnRNP G polypeptide in the ZW and WW GVs showed an exact match with the radioactive signal detected in the corresponding autoradiogram reflecting its binding to the WEc RNA probe. This binding was confirmed to be sequence-specific by the lack of hybridization signal with the PP6, BP7, BP7 RNA probes (data not shown). Occasionally and depending on the degree of resolution of the IEF in the basic pI region, three major closely migrating spots were found in the pI 10 area of the silver stained 2D-gels with ZW GVs instead of the two more frequently observed. The two more basic very close spots were of the same molecular mass while the third more acidic spot was of a lower molecular mass. The superimposition of the northwestern blot autoradiogram with the matching immunodetection blot showed that the WEc RNA probe bound the most basic polypeptide only (Figure 5B). We attempted to achieve a higher IEF resolution to determine whether the pI and molecular mass of the hnRNP G isoforms in the ZZ GVs were slightly different from those in the WW GVs. To this end we used the 2D DIGE fluorescence labelling method to reduce gel-to-gel variations. Samples of ZZ and WW GVs proteins were pre-labelled with different fluorescent dyes and co-migrated in the same gel. The superimposition of the fluorescent images did not reveal any difference for these isoforms between the two karyotypes (data not shown).Figure 5.


Differential RNA-binding activity of the hnRNP G protein correlated with the sex genotype in the amphibian oocyte.

Kanhoush R, Praseuth D, Perrin C, Chardard D, Vinh J, Penrad-Mobayed M - Nucleic Acids Res. (2011)

Interaction of the P. waltl hnRNP G isoforms with the WEc RNA. (A) 2D-NWA of the 32P-labelled RNA WEc probe with nuclear proteins from ZZ, ZW and WW GVs, followed by immunodetection using the anti-hnRNP G serum. Note that the homologues of hnRNPG formed a train of basic spots ranging from pI 9 to 10 with two major spots at pI 10. The most basic spot corresponded to the one detected by NWA in ZW and WW GVs. (B) An example of isoelectrofocusing of the protein extracts of the ZW GVs showing three spots of hnRNP G at pI 10 (arrowheads) in the silver stained gel (S). Note that only one spot was visible in the northwestern blot (N).
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Figure 5: Interaction of the P. waltl hnRNP G isoforms with the WEc RNA. (A) 2D-NWA of the 32P-labelled RNA WEc probe with nuclear proteins from ZZ, ZW and WW GVs, followed by immunodetection using the anti-hnRNP G serum. Note that the homologues of hnRNPG formed a train of basic spots ranging from pI 9 to 10 with two major spots at pI 10. The most basic spot corresponded to the one detected by NWA in ZW and WW GVs. (B) An example of isoelectrofocusing of the protein extracts of the ZW GVs showing three spots of hnRNP G at pI 10 (arrowheads) in the silver stained gel (S). Note that only one spot was visible in the northwestern blot (N).
Mentions: Northwestern and immunodetection assays performed in parallel on the same 2D western blot further confirmed the identification of the 42 kDa/pI 10 P. waltl polypeptides as hnRNPG homologues (Figure 5A). The position of the immunodetected spots corresponded to the polypeptides showed by mass spectrometry to be the homologues of the human hnRNPG protein. Furthermore, the western blot analysis revealed the existence in the oocytes of the three karyotypes, of a basic train of isoforms, ranging from pI 9 to 10 with closely similar molecular weights and present in different amounts. Three major spots were visible, one at pI 9 and the other two at pI 10, which could be distinguished from one another by a slight variation in molecular weight and pI. The superimposition of the autoradiogram with the western blot showed that none of the hnRNP G homologues bound the WEc RNA probe in the ZZ GVs because no radioactive hybridization signals were detected in the corresponding area of the autoradiogram. In contrast, the most basic hnRNP G polypeptide in the ZW and WW GVs showed an exact match with the radioactive signal detected in the corresponding autoradiogram reflecting its binding to the WEc RNA probe. This binding was confirmed to be sequence-specific by the lack of hybridization signal with the PP6, BP7, BP7 RNA probes (data not shown). Occasionally and depending on the degree of resolution of the IEF in the basic pI region, three major closely migrating spots were found in the pI 10 area of the silver stained 2D-gels with ZW GVs instead of the two more frequently observed. The two more basic very close spots were of the same molecular mass while the third more acidic spot was of a lower molecular mass. The superimposition of the northwestern blot autoradiogram with the matching immunodetection blot showed that the WEc RNA probe bound the most basic polypeptide only (Figure 5B). We attempted to achieve a higher IEF resolution to determine whether the pI and molecular mass of the hnRNP G isoforms in the ZZ GVs were slightly different from those in the WW GVs. To this end we used the 2D DIGE fluorescence labelling method to reduce gel-to-gel variations. Samples of ZZ and WW GVs proteins were pre-labelled with different fluorescent dyes and co-migrated in the same gel. The superimposition of the fluorescent images did not reveal any difference for these isoforms between the two karyotypes (data not shown).Figure 5.

Bottom Line: A proteomic approach has enabled the identification of an orthologue of the splicing factor hnRNP G in the amphibians Xenopus tropicalis, Ambystoma mexicanum, Notophthalmus viridescens and Pleurodeles walt, which shows a specific RNA-binding affinity similar to that of the human hnRN G protein.In situ hybridization to lampbrush chromosomes of P. waltl revealed the presence of a family of hnRNP G genes, which were mapped on the Z and W chromosomes and one autosome.This indicates that the isoforms identified in this study are possibly encoded by a gene family linked to the evolution of sex chromosomes similarly to the hnRNP G/RBMX gene family in mammals.

View Article: PubMed Central - PubMed

Affiliation: Institut Jacques Monod, UMR 7592, CNRS and Université Paris-Diderot, Museum National d'Histoire Naturelle, U 565, USM 503, UMR 5153, INSERM and CNRS, Paris, France.

ABSTRACT
A proteomic approach has enabled the identification of an orthologue of the splicing factor hnRNP G in the amphibians Xenopus tropicalis, Ambystoma mexicanum, Notophthalmus viridescens and Pleurodeles walt, which shows a specific RNA-binding affinity similar to that of the human hnRN G protein. Three isoforms of this protein with a differential binding affinity for a specific RNA probe were identified in the P. walt oocyte. In situ hybridization to lampbrush chromosomes of P. waltl revealed the presence of a family of hnRNP G genes, which were mapped on the Z and W chromosomes and one autosome. This indicates that the isoforms identified in this study are possibly encoded by a gene family linked to the evolution of sex chromosomes similarly to the hnRNP G/RBMX gene family in mammals.

Show MeSH
Related in: MedlinePlus