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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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Validation of the identification of hnRNP G and inter-species conservation of hnRNP G interaction with the WEc RNA. 1D-NWA of the binding of the 32P-labelled RNA WEc probe with nuclear proteins extracts from different species, followed by immunodetection using the canine anti-hnRNP G serum. (A) hnRNPG homologues in ZZ and ZW GVs of P. waltl. The 42 kDa labelled band (box) detected by NWA in ZW GVs corresponded exactly to the one detected by the anti-hnRNP G serum. Note that a band corresponding to hnRNP G was immunodetected in ZZ GVs, although no signal was visible in the corresponding blot after NWA. (B) hnRNP G from nuclear proteins extracts of human HeLa cells and GVs of the amphibians X. topicalis (X.t), N. viridescens (N.v) A. mexicanum (A.m) and P. waltl (P.w, ZW karyotype). Stars and circles indicate the bound hnRNP G.
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Figure 4: Validation of the identification of hnRNP G and inter-species conservation of hnRNP G interaction with the WEc RNA. 1D-NWA of the binding of the 32P-labelled RNA WEc probe with nuclear proteins extracts from different species, followed by immunodetection using the canine anti-hnRNP G serum. (A) hnRNPG homologues in ZZ and ZW GVs of P. waltl. The 42 kDa labelled band (box) detected by NWA in ZW GVs corresponded exactly to the one detected by the anti-hnRNP G serum. Note that a band corresponding to hnRNP G was immunodetected in ZZ GVs, although no signal was visible in the corresponding blot after NWA. (B) hnRNP G from nuclear proteins extracts of human HeLa cells and GVs of the amphibians X. topicalis (X.t), N. viridescens (N.v) A. mexicanum (A.m) and P. waltl (P.w, ZW karyotype). Stars and circles indicate the bound hnRNP G.

Mentions: In order to confirm the identification of the 42 kDa polypeptides as the homologues of the human hnRNP G protein, WEc RNA-binding assays followed by immunodetection assays with canine autoantibodies sera, which had been shown to react monospecifically with the human hnRNP G protein (26), were performed sequentially on blots of proteins from P. waltl GVs separated by mono-dimensional electrophoresis. No signal was immunodetected when the serum was pre-absorbed with the human hnRNP G (Supplementary Figure S1). In contrast, the canine anti-hnRNP G autoantibodies serum recognized one major labelled band in P. waltl GVs (Figure 4A). This band showed a perfect superimposition with the labelled 42 kDa band present in the corresponding northwestern blots with the ZW, WW GVs and Hela extracts (Figure 4A and B). In the case of the ZZ GVs an immunoreactive band was also detected at the same position but no radioactive signal was observed in the corresponding NWA. This result confirmed the presence of PwhnRNP G proteins in the 42 kDa band, which differentially bound the WEc RNA in the three P. waltl karyotypes.Figure 4.


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)

Validation of the identification of hnRNP G and inter-species conservation of hnRNP G interaction with the WEc RNA. 1D-NWA of the binding of the 32P-labelled RNA WEc probe with nuclear proteins extracts from different species, followed by immunodetection using the canine anti-hnRNP G serum. (A) hnRNPG homologues in ZZ and ZW GVs of P. waltl. The 42 kDa labelled band (box) detected by NWA in ZW GVs corresponded exactly to the one detected by the anti-hnRNP G serum. Note that a band corresponding to hnRNP G was immunodetected in ZZ GVs, although no signal was visible in the corresponding blot after NWA. (B) hnRNP G from nuclear proteins extracts of human HeLa cells and GVs of the amphibians X. topicalis (X.t), N. viridescens (N.v) A. mexicanum (A.m) and P. waltl (P.w, ZW karyotype). Stars and circles indicate the bound hnRNP G.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 4: Validation of the identification of hnRNP G and inter-species conservation of hnRNP G interaction with the WEc RNA. 1D-NWA of the binding of the 32P-labelled RNA WEc probe with nuclear proteins extracts from different species, followed by immunodetection using the canine anti-hnRNP G serum. (A) hnRNPG homologues in ZZ and ZW GVs of P. waltl. The 42 kDa labelled band (box) detected by NWA in ZW GVs corresponded exactly to the one detected by the anti-hnRNP G serum. Note that a band corresponding to hnRNP G was immunodetected in ZZ GVs, although no signal was visible in the corresponding blot after NWA. (B) hnRNP G from nuclear proteins extracts of human HeLa cells and GVs of the amphibians X. topicalis (X.t), N. viridescens (N.v) A. mexicanum (A.m) and P. waltl (P.w, ZW karyotype). Stars and circles indicate the bound hnRNP G.
Mentions: In order to confirm the identification of the 42 kDa polypeptides as the homologues of the human hnRNP G protein, WEc RNA-binding assays followed by immunodetection assays with canine autoantibodies sera, which had been shown to react monospecifically with the human hnRNP G protein (26), were performed sequentially on blots of proteins from P. waltl GVs separated by mono-dimensional electrophoresis. No signal was immunodetected when the serum was pre-absorbed with the human hnRNP G (Supplementary Figure S1). In contrast, the canine anti-hnRNP G autoantibodies serum recognized one major labelled band in P. waltl GVs (Figure 4A). This band showed a perfect superimposition with the labelled 42 kDa band present in the corresponding northwestern blots with the ZW, WW GVs and Hela extracts (Figure 4A and B). In the case of the ZZ GVs an immunoreactive band was also detected at the same position but no radioactive signal was observed in the corresponding NWA. This result confirmed the presence of PwhnRNP G proteins in the 42 kDa band, which differentially bound the WEc RNA in the three P. waltl karyotypes.Figure 4.

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