Xenbase Image ID: 152436

Fig. 6. Confirmation of newly discovered Ptbp1-controlled splicing events. A, agfg1, B, itga6, C, actn4, D, tpm4. Upper panels, genomic region encompassing the Ptbp1-regulated exons. Introns are represented as horizontal lines and exons as boxes. The exons are given arbitrary names, and their genomic coordinates are given in Table ST6. “a” stands for polyadenylation signal. The splice junctions are positioned along the gene structure. The junctions shown in black were not detected as differently used in control embryos and ptbp1 morphants, while the junctions shown in red and blue were detected as significantly (adjusted p<0.05) increased and decreased, respectively, in ptbp1 morphants. The exons in blue were detected as significantly (adjusted p<0.05) decreased in ptbp1 morphants. Lower panels, autoradiograms of representative RT-PCR experiments carried out with RNAs extracted from stage 28 embryos previously injected with the indicated molecules, and using the primers indicated by arrows in the upper panels. In all the experiments, the primers are designed to amplify both pseudo-alleles, and the forward primer is radiolabeled (*). The quantifications below the gels are means±s.d. of 3 independent experiments. A, agfg1 pre-mRNA contains a cassette exon (B), and its splicing was analysed with primers in flanking exons A and C. B, itga6 pre-mRNA contains a cassette exon (6A), and its splicing was analysed with primers in the constitutive flanking exons “const” and 6B. C, actn4 pre-mRNA contains a set of mutually exclusive exons (C and D), and its splicing was analysed with primers in flanking exons A and E, with Sac1 digestion before gel loading. D, tpm4 pre-mRNA contains two alternative terminal exons (E1 and E2), and its splicing was analysed with one forward primer in exons D and reverse primers in exons E1 and E2. Exons E2 of tpm4l and tpm4s differ by an indel located in the 3′UTR, explaining that the D-E2 amplimere is a doublet. The total amount of tpm4 mRNA was appraised from RT-PCR with primers in exons A and D, and eef1a1 (EF1α) confirmed similar RNA extractions. Image published in: Noiret M et al. (2017) Copyright © 2017. Image reproduced with permission of the Publisher, Elsevier B. V. Larger Image Printer Friendly View

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