Kanhoush R , Praseuth D , Perrin C , Chardard D , Vinh J , Penrad-Mobayed M .

	Figure 1. Pleurodeles waltl 42 kDa polypeptide(s) bind the WEc RNA specifically in the ZW and WW GVs. (A) Restriction map of the clone λ130 from which the RNA probes were derived by in vitro transcription. (B) NWA of 32P-labelled RNA probes with nuclear proteins from ZZ, ZW and WW GVs. Nuclear proteins were transferred to nitrocellulose membrane after electrophoresis in mono-dimensional polyacrylamide gels and incubated with the RNA-probes. Note that nuclear protein(s) in the 42 kDa range specifically bound to the WEc and PB6 RNA probes in ZW and WW karyotypes and not in ZZ karyotypes (arrow and box). No interaction could be detected for these proteins with the other RNA probes tested.
	Figure 2. Characterization of the WEc RNA-interacting 42 kDa polypeptides. (A) Six IEF-2D PAGE gels of ZZ, ZW and WW GVs proteins were prepared using 13 cm wide pH 6–11 IEF gel strips. IEF was followed by the separation in the second dimension, which was carried out simultaneously for the six gels. For each karyotype, one gel was processed for NWA using the WEc RNA as a probe, while the other was silver stained. Note that some spots (circles and double circles) were observed in the ZZ, ZW and WW northwestern blots. The one (double circles) was identified by LC-MS-MS as the DEAD (Asp-Glu-Ala-Asp) box polypeptide 17 (Homo sapiens, ATP-dependant RNA helicase DDX17) (Supplementary Table S3). In contrast, the 42 kDa and pI 10 spots (arrows) were observed in the ZW and WW and not in the ZZ northwestern blots although the corresponding polypeptides were visible in the stained gel. The spots of interest (42 kDa, pI 10) were cut out from the gel and processed for mass spectrometry. (B) High magnification of the areas corresponding to the spots of interest from the northwestern blots (ZZ, ZW and WW) and the stained gels (ZZ′, ZW′ and WW′).
	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.
	Figure 4. Validation of the identification of hnRNP G and inter-species conservation of hnRNP G interaction with the WEc RNA. 1D-NWA of the binding of the 32P-labelled RNA WEc probe with nuclear proteins extracts from different species, followed by immunodetection using the canine anti-hnRNP G serum. (A) hnRNPG homologues in ZZ and ZW GVs of P. waltl. The 42 kDa labelled band (box) detected by NWA in ZW GVs corresponded exactly to the one detected by the anti-hnRNP G serum. Note that a band corresponding to hnRNP G was immunodetected in ZZ GVs, although no signal was visible in the corresponding blot after NWA. (B) hnRNP G from nuclear proteins extracts of human HeLa cells and GVs of the amphibians X. topicalis (X.t), N. viridescens (N.v) A. mexicanum (A.m) and P. waltl (P.w, ZW karyotype). Stars and circles indicate the bound hnRNP G.
	Figure 5. Interaction of the P. waltl hnRNP G isoforms with the WEc RNA. (A) 2D-NWA of the 32P-labelled RNA WEc probe with nuclear proteins from ZZ, ZW and WW GVs, followed by immunodetection using the anti-hnRNP G serum. Note that the homologues of hnRNPG formed a train of basic spots ranging from pI 9 to 10 with two major spots at pI 10. The most basic spot corresponded to the one detected by NWA in ZW and WW GVs. (B) An example of isoelectrofocusing of the protein extracts of the ZW GVs showing three spots of hnRNP G at pI 10 (arrowheads) in the silver stained gel (S). Note that only one spot was visible in the northwestern blot (N).
	Figure 6. Relationship between PwhnRNPG phosphorylation state and RNA-binding activity. (A) Protein extracts of ZZ and WW GVs were separated in 2D-gels and stained with two sequential fluorescent dyes. Pro-Q Diamond (in blue) was specific of the phosphorylated proteins and Sypro Ruby (in red) revealed all proteins. A molecular mass indicator, PeppermintStick (PM), which contained phosphorylated proteins, β-casein (BC), ovalbumin (OA) and an non-phosphorylated protein, bovine serum albumin (BA) was run in parallel with each gel to show the specificity of the staining. The box indicates the major forms (a and b) of PwhnRNP G at pI 10. A high magnification of the merge of the two fluorescent stains is shown on the side of each gel. (B) Representative quantification of the phosphorylation degree of the major forms of PwhnRNP G (a and b) based on the intensity of their fluorescent staining by Pro-Q Diamond. Values were normalized relatively to the protein quantity estimated from the Sypro Ruby staining. (C) RNA-binding activity of dephosphorylated PwhnRNP G. Dephosphorylated ZZ (ZZd) and WW (WWd) or non-dephosphorylated (ZZ and WW) GV extracts were submitted to monodimensional NWA using WEc RNA probe followed by immunodetection (ID) of PwhnRNP G using the anti-hnRNP G serum. The arrow points to the PwhnRNPG protein. Note that a band corresponding to PwhnRNP G was immunodetected in ZZ GV, although no signal was visible in the corresponding blot after NWA in dephosphorylated or non-dephosphorylated extracts.
	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.

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