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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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Identification of the 42 kDa polypeptides as the hnRNPG homologues by Mass spectrometry analysis. (A) MALDI-TOF MS spectrum of one of the 42 kDa spots. The peaks are labelled with stars when the mass was identified as an hnRNP G peptide. (B) The peak at 1435.7 m/z was fragmented for further analysis. The N-to-C sequence of this peptide could be obtained directly by reading the spectrum from right to left (arrow). Thus, the sequence was 10LFIGGLNTETNEK22 and corresponded to an hnRNP G-specific peptide. Some a-, b-type and immonium fragments were also present and confirmed this unambiguous identification.
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Figure 3: Identification of the 42 kDa polypeptides as the hnRNPG homologues by Mass spectrometry analysis. (A) MALDI-TOF MS spectrum of one of the 42 kDa spots. The peaks are labelled with stars when the mass was identified as an hnRNP G peptide. (B) The peak at 1435.7 m/z was fragmented for further analysis. The N-to-C sequence of this peptide could be obtained directly by reading the spectrum from right to left (arrow). Thus, the sequence was 10LFIGGLNTETNEK22 and corresponded to an hnRNP G-specific peptide. Some a-, b-type and immonium fragments were also present and confirmed this unambiguous identification.

Mentions: In order to identify the 42 kDa polypeptide(s), GVs proteins from ZZ, ZW and WW oocytes were separated in duplicated bi-dimensional gels. One gel, to be used for preparative purposes, was stained to detect all proteins while the other was transferred to a nitrocellulose membrane, which was incubated with the WEc RNA probe (northwestern blot) and processed for autoradiography. Two pH ranges of immobilized gradients (IPG) were used for the first dimension. A wide pH range (pH 3–10) of IPGs was used initially, but no obvious difference in the pattern of protein spots labelled with the WEc probe was detected between the three types of GVs in NWA (data not shown). However, with a pH 6–11 gradient significant differences were found in the basic pI region. As shown in Figure 2, only one labelled polypeptide spot was detected in the 42 kDa/pI 10 region by the WEc RNA in the case of the ZW and WW GVs. This labelled spot was absent in the ZZ GVs extract contrary to other labelled spots of higher molecular mass, which were present for all three karyotypes, and are used as internal controls. Interestingly, extrapolation of the position of this 42 kDa/pI 10 spot to the matching silver-stained 2D-PAGE gels pointed to at least two closely migrating polypeptides that were present not only in the ZW and WW GVs but also in the ZZ GVs (Figure 2B, lower panels). Each of the corresponding spots was excised separately from the gels and submitted to mass spectrometry analysis. They were identified as homologues of the human hnRNP G/RBMX (accession number P38159) and the mouse hnRNP G (accession number O35479) proteins (Figure 3, Supplementary Tables S1 and S2).Figure 2.


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)

Identification of the 42 kDa polypeptides as the hnRNPG homologues by Mass spectrometry analysis. (A) MALDI-TOF MS spectrum of one of the 42 kDa spots. The peaks are labelled with stars when the mass was identified as an hnRNP G peptide. (B) The peak at 1435.7 m/z was fragmented for further analysis. The N-to-C sequence of this peptide could be obtained directly by reading the spectrum from right to left (arrow). Thus, the sequence was 10LFIGGLNTETNEK22 and corresponded to an hnRNP G-specific peptide. Some a-, b-type and immonium fragments were also present and confirmed this unambiguous identification.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3105392&req=5

Figure 3: Identification of the 42 kDa polypeptides as the hnRNPG homologues by Mass spectrometry analysis. (A) MALDI-TOF MS spectrum of one of the 42 kDa spots. The peaks are labelled with stars when the mass was identified as an hnRNP G peptide. (B) The peak at 1435.7 m/z was fragmented for further analysis. The N-to-C sequence of this peptide could be obtained directly by reading the spectrum from right to left (arrow). Thus, the sequence was 10LFIGGLNTETNEK22 and corresponded to an hnRNP G-specific peptide. Some a-, b-type and immonium fragments were also present and confirmed this unambiguous identification.
Mentions: In order to identify the 42 kDa polypeptide(s), GVs proteins from ZZ, ZW and WW oocytes were separated in duplicated bi-dimensional gels. One gel, to be used for preparative purposes, was stained to detect all proteins while the other was transferred to a nitrocellulose membrane, which was incubated with the WEc RNA probe (northwestern blot) and processed for autoradiography. Two pH ranges of immobilized gradients (IPG) were used for the first dimension. A wide pH range (pH 3–10) of IPGs was used initially, but no obvious difference in the pattern of protein spots labelled with the WEc probe was detected between the three types of GVs in NWA (data not shown). However, with a pH 6–11 gradient significant differences were found in the basic pI region. As shown in Figure 2, only one labelled polypeptide spot was detected in the 42 kDa/pI 10 region by the WEc RNA in the case of the ZW and WW GVs. This labelled spot was absent in the ZZ GVs extract contrary to other labelled spots of higher molecular mass, which were present for all three karyotypes, and are used as internal controls. Interestingly, extrapolation of the position of this 42 kDa/pI 10 spot to the matching silver-stained 2D-PAGE gels pointed to at least two closely migrating polypeptides that were present not only in the ZW and WW GVs but also in the ZZ GVs (Figure 2B, lower panels). Each of the corresponding spots was excised separately from the gels and submitted to mass spectrometry analysis. They were identified as homologues of the human hnRNP G/RBMX (accession number P38159) and the mouse hnRNP G (accession number O35479) proteins (Figure 3, Supplementary Tables S1 and S2).Figure 2.

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