4118 Nucleic Acids Research, 2011, Vol. 39, No. 10 dephosphorylated form of PwhnRNPG in WW GV still interacted with the WEc RNA, as shown on 1D-NWA while that of ZZ GV did not (Figure 6C) These data demonstrated that hnRNP G phosphorylation was not necessary for its interaction with the WEc RNA. Evidence for a PwhnRNPG multigene family in P. waltl 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 tran- scripts 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 hybrid- izing 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 chromo- some (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 hybrid- ization 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 respect- ively 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). DISCUSSION The PwhnRNP G protein shows a sequence-speciﬁc RNA-binding activity associated with the presence of the W chromosome in the oocyte genotype hnRNP G is a sequence-speciﬁc RNA-binding protein known to be implicated in the splicing of pre-mRNAs. Using an RNA probe that we had previously shown to interact with the human hnRNP G via a sequence-speciﬁc motif (21), we provide evidence for the presence of several PwhnRNP G isoforms in the ZZ, ZW and WW oocytes of 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 mm.