4110 Nucleic Acids Research, 2011, Vol. 39, No. 10 contains an early stop codon in its sequence (12). The RBMY and hnRNP G-T proteins are expressed exclusive- ly in the male germinal cells in the testis and are implicated in the splicing of testis-speciﬁc exons (15,16). Although the RBMY and hnRNP G-T proteins show 59 and 74% similarity with hnRNP G, respectively, they exhibit differ- ent RNA-binding afﬁnities. RNA motifs recognized with high afﬁnity by the RBMY and hnRNP G-T proteins are not bound by the hnRNP G protein (17,18). It has been shown that CCC/A-rich RNAs are preferentially bound by the N-terminal RNA-binding domain (N-ter RRM) of hnRNP G (19). We have reported recently that a stem-(G GAAA)-loop RNA motif present in the WEc RNA, a 250 nt RNA substrate, formerly called BP6 RNA (20), is a speciﬁc binding site for a newly identiﬁed C-terminal RNA-binding domain of hnRNP G (21). Altogether, these data suggest that hnRNP G is a sequence-speciﬁc binding protein, which recognizes cis-acting RNA elements in its targets. In Pleurodeles waltl where the female is heterogametic (ZW) and the male is homogametic (ZZ), functionally phenotypic ZZ and WW females can be experimentally produced (22,23). This enabled us to develop a proteomic approach with the aim of identifying RNA-binding proteins (RBPs) encoded by genes linked to the sex chromosomes. We have previously shown that the WEc RNA labelled speciﬁcally the W chromosome by binding to RBPs associated with transcription units located on three adjacent lampbrush loops in nuclear spreads of P. waltl oocytes (20). In the present work, we have used the WEc RNA as a probe to show the presence in the P. waltl oocyte of several isoforms of hnRNP G, referred to as PwhnRNP G, whose differential RNA afﬁnity was correlated with the presence of the Z or W sex chromosomes. We also obtained evidence for a PwhnRNP G multigene family whose members are located on the Z and W sex chromosomes and an autosome in P. waltl. EXPERIMENTAL PROCEDURES Oocytes and cell culture Ovarian biopsies were performed on adult females P. waltl anesthetized in 0.15% tricaine methane sulfonate (MS222, Sigma Chemical, St Louis, MO, USA). Oocytes were defolliculated for 2–3 h in saline buffer OR2 containing 0.15% collagenase type II (Sigma Chemical). Stage IV–VI oocytes were selected and main- tained in MBS medium (24) at 18 C. Human cervical carcinoma cells HeLa (ATCC CCL-2) were cultured in RPMI 1640 medium (GIBCO, Invitrogen) supplemented with 5% (v/v) fetal bovine serum and 2 mM L-glutamine. Cell cultures were maintained at 37 C in 5% CO2 atmosphere. Nuclear extracts Oocytes nuclear extracts. Oocytes were incubated in MBS containing 2% TCA (w/v) for 15–20 min at 4 C to pre- cipitate proteins, washed several times in cold MBS and kept at 4 C up to 3 days before being individually dissected in MBS. Germinal vesicles (GVs) were isolated, transferred to microtubes, washed in water, then in 70% ethanol. Air-dried GVs were resuspended in the denaturing electrophoresis loading buffer. HeLa cells nuclear extracts. Cells were washed twice with cold PBS and resuspended in buffer A: 10 mM Tris pH 8.0, 3 mM MgCl2, 25 mM NaCl, protease inhibitor (Complete Protease Inhibitor Cocktail Tablets, Roche Applied science). After 2 min incubation on ice, cells were disrupted using an Ultra-Turrax homogenizer and centrifuged at 1200g for 5 min. Nuclei were resuspended in buffer B: 0.25 M Sucrose, 10 mM Tris pH 8.0, 10 mM MgCl2, 10 mM NaCl, protease inhibitor cocktail. The sus- pension was layered over modiﬁed buffer B (0.88 M Sucrose) and centrifuged for 10 min. Nuclei were resus- pended in the denaturing electrophoresis loading buffer. Gel electrophoresis and staining For mono-dimensional electrophoresis, samples were denatured by heating in Laemmli buffer for 5 min at 95 C and loaded on a 10% SDS–PAGE gel (Mini PROTEAN II or Protean II xi from Bio-rad). For bi-dimensional electrophoresis, samples were resuspended in DeStreak rehydration solution (8 M urea, 1 M thiourea, 2.5% CHAPS, hydroxyethyl disulﬁde, GE Healthcare) plus 1% carrier ampholytes mixture PH 6-11 (GE Healthcare). The isoelectric focusing (IEF) conditions were optimized for focalization of basic proteins. After 12 h of strip rehydration, samples were loaded on the anodic side of 13 cm strips that had a linear pH 6–11 gradient (IPG strips, GE Healthcare). The IEF was per- formed on an Ettan IPGphor (GE Healthcare) at 20 C using the following parameters: voltage was increased gradually to 500 V for 100 Vh then to 4000 V for 3400 Vh and ﬁnally to 8000 V for 13 330 Vh. Prior to the second dimension the strips were equilibrated in 6 M urea, 2% SDS, 1% DTT and 30% glycerol. In a second equili- bration step the DTT was replaced by iodoacetamide to prevent proteins reoxidation. The second dimension was performed in Ettan Dalt VI (GE Healthcare) or Protean II xi (Bio-rad) vertical electrophoresis apparatuses using 12% SDS–PAGE gels. 2D gels destined for phosphopro- tein detection were sequentially stained with ﬂuorescent dyes Pro-Q Diamond and SYPRO Ruby (Molecular Probes, Invitrogen) according to manufacturer’s proto- cols. The Pro-Q diamond stain allows in-gel detection of phosphate groups attached to tyrosine, serine or threonine residues. For individual phosphoproteins, the strength of the signal correlates with the number of phosphate groups and with the protein quantity. The SYPRO Ruby stain is a quantitative stain that reveals all the proteins. The molecular mass indicator PeppermintStick (Molecular Probes, Invitrogen) was used to show the speciﬁcity of the Pro-Q diamond dye. The ﬂuorescent gels images were obtained on a Typhoon Imaging System 9200 or 9400 (GE Healthcare). Nuclear extracts destined to alkaline phosphatase treat- ment were prepared as follows: aliquots of 60 GVs were extracted by homogenization at 4 C in lysis buffer