P. waltl whose differential afﬁnity for this RNA was correlated with the presence of the W chromosome. One of these isoforms in the ZW and WW GVs bound to the WEc RNA, while none of the isoforms in the ZZ oocytes bound to this RNA. Because the molecular weights and the isoelectric points of these isoforms are very close in the ZZ, ZW and WW GVs, it can be assumed that RNA- binding domains are present in the ZZ isoforms but display a binding afﬁnity for RNA sequences different from the ones that we have used as probes. It has been shown that a small divergence in the amino acid compos- ition of an RNA-binding domain can have a great effect on its afﬁnity for RNAs, as it is the case for the divergence of the N-ter RRM between the human RBMY protein and its mouse orthologue mRBMY (17). The human RBMY RRM recognizes with high afﬁnity stem-loop- structured RNAs that contain the sequence CA/UCAA. Because of a difference of two critical amino acids, the mRBMY RRM is unable to bind efﬁciently to these RNAs. Therefore, it is possible that a small sequence divergence in the ZZ isoforms or a differential PTM is responsible for their lack of afﬁnity towards the WEc RNA. A multigenic RBMZ/RBMW family in amphibians We also provide evidence for the existence of a multigenic family of PwhnRNP G genes in P. waltl. hnRNP G tran- scripts were mapped by in situ hybridization to at least three distinct genomic loci on different chromosomes: RBMZ on the Z chromosome, RBMW on the W chromo- some and RBMA on the autosomal bivalent VI. No P. waltl genomic sequences are available to test this as- sumption. A single gene coding for the hnRNP G protein was found in X. tropicalis cDNAs databases (35,36). However, the possibility that other copies of this gene are present in the X. tropicalis genome cannot be ruled out because low abundance mRNAs are often poorly rep- resented in databases. It is also possible that this gene family has evolved differently in P. waltl and X. tropicalis. The proposed P. waltl RBMZ/RBMW multigene family does not necessarily reﬂect a general organization of the hnRNP G-encoding genes in other amphibian genomes. Although sex chromosomes have been intensively studied in mammals, very little is known about the origin of sex chromosomes in amphibians. Genes linked to sex chromosomes could have evolved differently in very closely related amphibians. A recent study proposed that chromosome III is the W chromosome in X. laevis because it carries the DM-W gene, a candidate for the female-determining gene in this species (37). However, no DM-W orthologue was found in the X. tropicalis genome (38). Furthermore, eight genes necessary for sex differenti- ation have different chromosomal locations in X. laevis, X. tropicalis and Rana rugosa suggesting that the W chromosome was acquired independently in X. laevis (38). Correlation between hnRNP G isoforms and RBMX-like genes Our results raise the question of the relation between the hnRNP G isoforms and the multiple RBMX-like genes Nucleic Acids Research, 2011, Vol. 39, No. 10 4119 localized on different chromosomes. At least two possibilities can be considered. The ﬁrst one is that the RBMZ, RBMW and RBMA genes code for different proteins, RBMZ, RBMW and RBMA. At pI 10, we observed consistently two major hnRNP G isoforms in the ZW, ZZ and WW GVs and occasionally three isoforms in the ZW oocytes, which differed slightly in their pIs, the more basic isoform being the most abundant. We propose that the acidic isoform could cor- respond to the RBMA protein encoded by the autosomal RBMA gene because it is common to the three ZZ, ZW and ZW karyotypes; the basic isoform could correspond to two very closely related proteins, RBMZ and RBMW encoded by separate genes, the RBMW protein being the one binding to the WEc RNA. An alternative possibility is that these proteins are the products of a single gene, the RBMA autosomal gene, the RBMZ and RBMW genes being non-functional because of frameshifts or stop codons in their coding sequences. The different pI isoforms could represent splicing variants or result from post-translational modiﬁcations regulated by one or several W-linked genes. Phosphorylation is one of the most currently observed modiﬁcations of the proteins that regulate their functions. Indeed we noted a signiﬁcant difference in the phosphorylation level between the Pleurodeles hnRNP G isoforms. However, dephospho- rylation of hnRNP G in ZZ or WW GV extracts did not affect their sequence-speciﬁc binding to this substrate. Other modiﬁcations of the protein such as methylation and acetylation that can affect the RNA-binding afﬁnity could also be considered. Regardless of their origin, it is clear that the PwhnRNP G isoforms show differential RNA-binding activity. Therefore, we can assume that their RNA afﬁnity is directly or indirectly, under the control of the sex genotype, because the only variant that recognizes the WEc RNA is found in the ZW and WW karyotypes exclusively. This study demonstrates that the proteomic approach can yield important information concerning differential RNA-binding afﬁnities of RBP isoforms correlated with the sex genotype in animal species not currently subjected to genomic studies. Our ﬁndings open the door for further investigations of the role of the hnRNP G gene family in the regulation of RNA processing in a sequence-speciﬁc manner in non-mammalian vertebrates. SUPPLEMENTARY DATA Supplementary Data are available at NAR Online. ACKNOWLEDGEMENTS We are grateful to Jean-Antoine Lepesant for a careful review of the article and helpful discussion. We thank Reiner Veitia for critically reading the article and Franc¸ ois Guillonneau for its advices for the alkaline protein separation. We thank Vale´ rie Labas for her con- tribution to the Mass spectrometry analysis and Ce´ dric Absalon and Delphine Fichelle for their contribution at