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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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Relationship between PwhnRNPG phosphorylation state and RNA-binding activity. (A) Protein extracts of ZZ and WW GVs were separated in 2D-gels and stained with two sequential fluorescent dyes. Pro-Q Diamond (in blue) was specific of the phosphorylated proteins and Sypro Ruby (in red) revealed all proteins. A molecular mass indicator, PeppermintStick (PM), which contained phosphorylated proteins, β-casein (BC), ovalbumin (OA) and an non-phosphorylated protein, bovine serum albumin (BA) was run in parallel with each gel to show the specificity of the staining. The box indicates the major forms (a and b) of PwhnRNP G at pI 10. A high magnification of the merge of the two fluorescent stains is shown on the side of each gel. (B) Representative quantification of the phosphorylation degree of the major forms of PwhnRNP G (a and b) based on the intensity of their fluorescent staining by Pro-Q Diamond. Values were normalized relatively to the protein quantity estimated from the Sypro Ruby staining. (C) RNA-binding activity of dephosphorylated PwhnRNP G. Dephosphorylated ZZ (ZZd) and WW (WWd) or non-dephosphorylated (ZZ and WW) GV extracts were submitted to monodimensional NWA using WEc RNA probe followed by immunodetection (ID) of PwhnRNP G using the anti-hnRNP G serum. The arrow points to the PwhnRNPG protein. Note that a band corresponding to PwhnRNP G was immunodetected in ZZ GV, although no signal was visible in the corresponding blot after NWA in dephosphorylated or non-dephosphorylated extracts.
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Figure 6: Relationship between PwhnRNPG phosphorylation state and RNA-binding activity. (A) Protein extracts of ZZ and WW GVs were separated in 2D-gels and stained with two sequential fluorescent dyes. Pro-Q Diamond (in blue) was specific of the phosphorylated proteins and Sypro Ruby (in red) revealed all proteins. A molecular mass indicator, PeppermintStick (PM), which contained phosphorylated proteins, β-casein (BC), ovalbumin (OA) and an non-phosphorylated protein, bovine serum albumin (BA) was run in parallel with each gel to show the specificity of the staining. The box indicates the major forms (a and b) of PwhnRNP G at pI 10. A high magnification of the merge of the two fluorescent stains is shown on the side of each gel. (B) Representative quantification of the phosphorylation degree of the major forms of PwhnRNP G (a and b) based on the intensity of their fluorescent staining by Pro-Q Diamond. Values were normalized relatively to the protein quantity estimated from the Sypro Ruby staining. (C) RNA-binding activity of dephosphorylated PwhnRNP G. Dephosphorylated ZZ (ZZd) and WW (WWd) or non-dephosphorylated (ZZ and WW) GV extracts were submitted to monodimensional NWA using WEc RNA probe followed by immunodetection (ID) of PwhnRNP G using the anti-hnRNP G serum. The arrow points to the PwhnRNPG protein. Note that a band corresponding to PwhnRNP G was immunodetected in ZZ GV, although no signal was visible in the corresponding blot after NWA in dephosphorylated or non-dephosphorylated extracts.

Mentions: The theorical isoelectric point of the human hnRNPG predicted from its primary protein sequence is 10.06. As shown in Figure 5A, the 2D migration profile of its immunodetected homologues in P. waltl oocytes extracts showed a train of spots with nearly identical molecular weights and isoelectric points ranging between pI 9 and pI 10. These pI shifts could result from post-translation modifications (PTMs) such as phosphorylation, acetylation or methylation, which are known to play a significant role in the regulation of gene expression by modifying protein–protein and/or nucleic acids–proteins interactions. Phosphorylation is one of the most common PTM that can be detected by a direct in-gel method. Because the hnRNP G isoforms of the ZZ GVs did not bind the WEc RNA probe and differed in their ability to bind the WEc RNA probe in the WW GVs, we investigated the phosphorylation state of these proteins, which may influence their RNA affinity. Pro-Q diamond phosphoprotein stain was used on 2D gels for a sensitive and direct in-gel detection of phosphoproteins irrespective of the phosphoamino acid (‘Materials and Methods’ section). The subsequent staining of the same 2D-gels using the total-protein SYPRO Ruby stain provided a measure of the phosphorylation level relative to the total amount of the protein. As seen in Figure 6, the two major spots corresponding to hnRNP G appeared as phosphorylated in ZZ and WW GVs. Nevertheless, the degree of phosphorylation of the WW-b isoform spot, which bound the WEc RNA probe, was lower than that of the WW-a, ZZ-a and ZZ-b isoforms, which did not bind this probe.Figure 6.


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)

Relationship between PwhnRNPG phosphorylation state and RNA-binding activity. (A) Protein extracts of ZZ and WW GVs were separated in 2D-gels and stained with two sequential fluorescent dyes. Pro-Q Diamond (in blue) was specific of the phosphorylated proteins and Sypro Ruby (in red) revealed all proteins. A molecular mass indicator, PeppermintStick (PM), which contained phosphorylated proteins, β-casein (BC), ovalbumin (OA) and an non-phosphorylated protein, bovine serum albumin (BA) was run in parallel with each gel to show the specificity of the staining. The box indicates the major forms (a and b) of PwhnRNP G at pI 10. A high magnification of the merge of the two fluorescent stains is shown on the side of each gel. (B) Representative quantification of the phosphorylation degree of the major forms of PwhnRNP G (a and b) based on the intensity of their fluorescent staining by Pro-Q Diamond. Values were normalized relatively to the protein quantity estimated from the Sypro Ruby staining. (C) RNA-binding activity of dephosphorylated PwhnRNP G. Dephosphorylated ZZ (ZZd) and WW (WWd) or non-dephosphorylated (ZZ and WW) GV extracts were submitted to monodimensional NWA using WEc RNA probe followed by immunodetection (ID) of PwhnRNP G using the anti-hnRNP G serum. The arrow points to the PwhnRNPG protein. Note that a band corresponding to PwhnRNP G was immunodetected in ZZ GV, although no signal was visible in the corresponding blot after NWA in dephosphorylated or non-dephosphorylated extracts.
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Related In: Results  -  Collection

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Figure 6: Relationship between PwhnRNPG phosphorylation state and RNA-binding activity. (A) Protein extracts of ZZ and WW GVs were separated in 2D-gels and stained with two sequential fluorescent dyes. Pro-Q Diamond (in blue) was specific of the phosphorylated proteins and Sypro Ruby (in red) revealed all proteins. A molecular mass indicator, PeppermintStick (PM), which contained phosphorylated proteins, β-casein (BC), ovalbumin (OA) and an non-phosphorylated protein, bovine serum albumin (BA) was run in parallel with each gel to show the specificity of the staining. The box indicates the major forms (a and b) of PwhnRNP G at pI 10. A high magnification of the merge of the two fluorescent stains is shown on the side of each gel. (B) Representative quantification of the phosphorylation degree of the major forms of PwhnRNP G (a and b) based on the intensity of their fluorescent staining by Pro-Q Diamond. Values were normalized relatively to the protein quantity estimated from the Sypro Ruby staining. (C) RNA-binding activity of dephosphorylated PwhnRNP G. Dephosphorylated ZZ (ZZd) and WW (WWd) or non-dephosphorylated (ZZ and WW) GV extracts were submitted to monodimensional NWA using WEc RNA probe followed by immunodetection (ID) of PwhnRNP G using the anti-hnRNP G serum. The arrow points to the PwhnRNPG protein. Note that a band corresponding to PwhnRNP G was immunodetected in ZZ GV, although no signal was visible in the corresponding blot after NWA in dephosphorylated or non-dephosphorylated extracts.
Mentions: The theorical isoelectric point of the human hnRNPG predicted from its primary protein sequence is 10.06. As shown in Figure 5A, the 2D migration profile of its immunodetected homologues in P. waltl oocytes extracts showed a train of spots with nearly identical molecular weights and isoelectric points ranging between pI 9 and pI 10. These pI shifts could result from post-translation modifications (PTMs) such as phosphorylation, acetylation or methylation, which are known to play a significant role in the regulation of gene expression by modifying protein–protein and/or nucleic acids–proteins interactions. Phosphorylation is one of the most common PTM that can be detected by a direct in-gel method. Because the hnRNP G isoforms of the ZZ GVs did not bind the WEc RNA probe and differed in their ability to bind the WEc RNA probe in the WW GVs, we investigated the phosphorylation state of these proteins, which may influence their RNA affinity. Pro-Q diamond phosphoprotein stain was used on 2D gels for a sensitive and direct in-gel detection of phosphoproteins irrespective of the phosphoamino acid (‘Materials and Methods’ section). The subsequent staining of the same 2D-gels using the total-protein SYPRO Ruby stain provided a measure of the phosphorylation level relative to the total amount of the protein. As seen in Figure 6, the two major spots corresponding to hnRNP G appeared as phosphorylated in ZZ and WW GVs. Nevertheless, the degree of phosphorylation of the WW-b isoform spot, which bound the WEc RNA probe, was lower than that of the WW-a, ZZ-a and ZZ-b isoforms, which did not bind this probe.Figure 6.

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