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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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In situ hybridization to nascent RNA transcripts on lampbrush chromosomes from P. waltl using the 35S-labelled RBMX cRNA. (A) Autoradiographs of bivalents VI and IV (ZW and WW). (B) High magnification of the boxed area of the A autoradiograph. Arrows point to the labelled loops. On bivalent VI, each of the two hybridization sites concerns a uniformly labelled pair of loops (arrows). On ZW bivalent, strongly and uniformly labelled loops are observed on only one homologue (arrows). On bivalent WW, each of the two hybridization sites concerns a partially and lightly labelled pair of loops (arrows). Scale bars: 50 µm.
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Figure 7: In situ hybridization to nascent RNA transcripts on lampbrush chromosomes from P. waltl using the 35S-labelled RBMX cRNA. (A) Autoradiographs of bivalents VI and IV (ZW and WW). (B) High magnification of the boxed area of the A autoradiograph. Arrows point to the labelled loops. On bivalent VI, each of the two hybridization sites concerns a uniformly labelled pair of loops (arrows). On ZW bivalent, strongly and uniformly labelled loops are observed on only one homologue (arrows). On bivalent WW, each of the two hybridization sites concerns a partially and lightly labelled pair of loops (arrows). Scale bars: 50 µm.

Mentions: In order to determine whether the different PwhnRNP G isoforms expressed in the Pleurodeles oocyte corresponded to proteins coded by one or several genes, we carried out in situ hybridization of an RBMX cRNA probe to RNA transcripts of oocyte lampbrush chromosomes (LBCs). In situ hybridization of specific probes to nascent transcripts of lateral loops of LBCs is a powerful approach to tackle this question in amphibians. It yields strong signals due to the binding of the probe to many closely-packed RNA transcripts in these loops [for review, see (27,29,30)]. The chromosomal localization of these hybridizing loops can be precisely determined because LBCs maps were established in many amphibian species thanks to the presence of prominent landmarks, which show a distinctive morphology and are reproducibly observed at constant sites along the axis of each chromosome (31–33). This is well illustrated in the case of P. waltl where the ZW sex bivalent chromosomes (bivalent IV) are easily identifiable in the oocyte karyotype contrary to the mitotic karyotype in which sex chromosomes cannot be distinguished (34,20). In addition, the hybridization to the Z or to the W chromosomes can be further confirmed using ZZ or WW GVs. Because the sequence of the hnRNP G/RBMX cDNA is highly conserved among vertebrate species (87% of similarity between X. tropicalis and human and 97% of similarity between X. tropicalis and X. laevis, Supplementary Figure S2), two antisense RBMX RNAs from X. tropicalis were used as probes. Results clearly indicated that RBMX gene expression took place at the level of at least three distinct genomic sites: one on the autosome VI, one on the sex chromosome Z and one on the sex chromosome W (Figure 7). We have named these genes RBMA, RBMZ and RBMW respectively for RBM linked to an autosome or to the Z or W sex chromosomes. As shown in Figure 7, the labelling signal on the Z chromosome was stronger than that observed on the W chromosome, possibly because of the presence of multiple gene copies expressed on at least two adjacent loops. These results suggested that in P. waltl the PwhnRNP G protein is encoded by a multigenic family, which we referred to as the RBMZ/RBMW family by analogy with the RBMX/RBMY family in mammals. This family would be comprised of several genes located on the Z chromosome (RBMZs), the W chromosome (RBMW(s) and at least one autosome, chromosome VI, (RBMA).Figure 7.


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)

In situ hybridization to nascent RNA transcripts on lampbrush chromosomes from P. waltl using the 35S-labelled RBMX cRNA. (A) Autoradiographs of bivalents VI and IV (ZW and WW). (B) High magnification of the boxed area of the A autoradiograph. Arrows point to the labelled loops. On bivalent VI, each of the two hybridization sites concerns a uniformly labelled pair of loops (arrows). On ZW bivalent, strongly and uniformly labelled loops are observed on only one homologue (arrows). On bivalent WW, each of the two hybridization sites concerns a partially and lightly labelled pair of loops (arrows). Scale bars: 50 µm.
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Figure 7: In situ hybridization to nascent RNA transcripts on lampbrush chromosomes from P. waltl using the 35S-labelled RBMX cRNA. (A) Autoradiographs of bivalents VI and IV (ZW and WW). (B) High magnification of the boxed area of the A autoradiograph. Arrows point to the labelled loops. On bivalent VI, each of the two hybridization sites concerns a uniformly labelled pair of loops (arrows). On ZW bivalent, strongly and uniformly labelled loops are observed on only one homologue (arrows). On bivalent WW, each of the two hybridization sites concerns a partially and lightly labelled pair of loops (arrows). Scale bars: 50 µm.
Mentions: In order to determine whether the different PwhnRNP G isoforms expressed in the Pleurodeles oocyte corresponded to proteins coded by one or several genes, we carried out in situ hybridization of an RBMX cRNA probe to RNA transcripts of oocyte lampbrush chromosomes (LBCs). In situ hybridization of specific probes to nascent transcripts of lateral loops of LBCs is a powerful approach to tackle this question in amphibians. It yields strong signals due to the binding of the probe to many closely-packed RNA transcripts in these loops [for review, see (27,29,30)]. The chromosomal localization of these hybridizing loops can be precisely determined because LBCs maps were established in many amphibian species thanks to the presence of prominent landmarks, which show a distinctive morphology and are reproducibly observed at constant sites along the axis of each chromosome (31–33). This is well illustrated in the case of P. waltl where the ZW sex bivalent chromosomes (bivalent IV) are easily identifiable in the oocyte karyotype contrary to the mitotic karyotype in which sex chromosomes cannot be distinguished (34,20). In addition, the hybridization to the Z or to the W chromosomes can be further confirmed using ZZ or WW GVs. Because the sequence of the hnRNP G/RBMX cDNA is highly conserved among vertebrate species (87% of similarity between X. tropicalis and human and 97% of similarity between X. tropicalis and X. laevis, Supplementary Figure S2), two antisense RBMX RNAs from X. tropicalis were used as probes. Results clearly indicated that RBMX gene expression took place at the level of at least three distinct genomic sites: one on the autosome VI, one on the sex chromosome Z and one on the sex chromosome W (Figure 7). We have named these genes RBMA, RBMZ and RBMW respectively for RBM linked to an autosome or to the Z or W sex chromosomes. As shown in Figure 7, the labelling signal on the Z chromosome was stronger than that observed on the W chromosome, possibly because of the presence of multiple gene copies expressed on at least two adjacent loops. These results suggested that in P. waltl the PwhnRNP G protein is encoded by a multigenic family, which we referred to as the RBMZ/RBMW family by analogy with the RBMX/RBMY family in mammals. This family would be comprised of several genes located on the Z chromosome (RBMZs), the W chromosome (RBMW(s) and at least one autosome, chromosome VI, (RBMA).Figure 7.

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