Méreau A , Anquetil V , Lerivray H , Viet J , Schirmer C , Audic Y , Legagneux V , Hardy S , Paillard L .

	FIG 1 Coexpression of ptbp1 and esrp1 in Xenopus embryos. (A) Phylogenetic tree showing the distances between Xenopus esrp1 (Xenopus laevis and Xenopus tropicalis), human (Homo sapiens) ESRP1 and ESRP2, and rodent (Rattus norvegicus and Mus musculus) Esrp1 and Esrp2. (B) (Top left) Schematic drawing of tpm1a pre-mRNA. Constitutive exon 8 is spliced either to terminal exon 9A9′ to produce the α7 isoform or to terminal exon 9D to produce the O5 isoform. An additional exon, 9B, was omitted for clarity. The arrows reveal the positions of the PCR primers. (Right) The somites and the epidermis were dissected from stage 30 Xenopus embryos. The relative abundances of the indicated isoforms of the tpm1a mRNAs and of the eef1a1 mRNA (EF1α, as a loading control) were estimated in the two fractions and total nondissected sibling embryos by radioactive semiquantitative RT-PCR. (Lower left) Quantification from 3 similar experiments (mean ± SD). (C and D) The relative abundances of Ptbp1, Esrp1, and Pcna (as a loading control) in dissected embryos (C) or in total embryos arrested at different stages of development (D) were measured by Western blotting. The blastula is stages 6 to 8, the gastrula is stages 9 to 11, the neurula is stage 15, and the tail bud is stages 28 to 30 (31). In all panels, the positions of the molecular mass markers are indicated on the right of the gels.
	FIG 2 The knockdown of esrp1 reduces the abundance of the Ptbp1 protein. (A) An mRNA encoding a V5-tagged version of Esrp1 with the wild-type (WT) 5′ region (WT-esrp1-V5) was injected with either a control (ctrl) or the esrp1 MO in two-cell embryos. The embryos were allowed to develop until stage 15 (neurula) before protein extraction and Western blot analysis using anti-V5 and anti-Pcna (loading control) antibodies. (B) The indicated morpholinos were injected in both blastomeres of two-cell embryos. The embryos were allowed to develop until stages 28 to 30 (tail bud) before protein extraction and Western blot analysis. (C) An mRNA encoding a V5-tagged version of Ptbp1 with the wild-type 5′ region (WT-ptbp1-V5) was injected alone or in combination with the esrp1 MO in both blastomeres of two-cell embryos. The embryos were allowed to develop until stages 28 to 30 before protein extraction for Western blot analysis using anti-Ptbp1, anti-V5, and anti-Pcna (loading control) antibodies. Endo., endogenous. (D) esrp1 MO and/or an mRNA encoding a V5-tagged version of Esrp1 immune to the esrp1 MO (esrp1-V5 mRNA) was injected in both blastomeres of two-cell embryos. The embryos were allowed to develop until stage 15 before protein extraction and Western blot analysis. (E) The indicated morpholinos were injected in both blastomeres of two-cell embryos. The embryos were allowed to develop until stage 35 (tadpole), and the presence of blisters was recorded. (Left) Representative embryos (arrows point to the blisters); (right) quantification of the blister phenotype. (F) An mRNA encoding a V5-tagged version of Ptbp1 (which is immune to the ptbp1 MO) and/or the indicated morpholinos were injected in both blastomeres of two-cell embryos. The embryos were allowed to develop until stages 28 to 30. The total RNAs were extracted and quantified by semiquantitative RT-PCR with a forward primer hybridizing to the constitutive exon 8 and a reverse primer hybridizing to the nonmuscular exon 9D of tpm1a mRNA, to amplify the nonmuscular (O5) isoform (Fig. 1B). Primers specific for eef1a1 mRNAs were also used. (Left) Results of a representative experiment; (right) quantification from 3 independent experiments (mean ± SD). The tpm1a-O5/eef1a1 ratios were normalized by the ratio in the noninjected embryos in each experiment to correct for the different specific activities of the radiolabeled primers. (G) Quantification by real-time PCR of ptbp2 mRNA abundance relative to eef1a1 mRNA abundance in embryos injected with the same molecules used for the assay whose results are presented in panel F (mean ± SD from 3 independent experiments).
	FIG 3 The knockdown of esrp1 reduces the abundance of ptbp1 mRNA but not that of ptbp1 pre-mRNA. (A) Positions of the primers in ptbp1 RNA used for real-time PCR. (B) Both blastomeres of two-cell embryos were injected with the esrp1 MO or left noninjected (NI). The development of the embryos was arrested at stages 28 to 30 for RNA extraction. The abundance of ptbp1 mRNA relative to that of eef1a1 mRNA was determined by real-time RT-PCR (mean ± SD from 5 independent experiments). Primers hybridizing to the indicated exons (identified by the prefix e on the x axis) were used. The P values of Student's t tests are given above the bars. (C) Same as panel B, except that pairs of primers directed against the indicated intron (i)-exon (e) junctions were used to amplify ptbp1 pre-mRNA. No amplification occurred in control experiments performed without reverse transcriptase.
	FIG 4 Self-regulatory feedback loop of ptbp1 expression in Xenopus epidermis. The ptbp1 MO or an mRNA encoding a V5-tagged version of Ptbp1 protein (ptbp1-V5 mRNA) was injected in both blastomeres of two-cell embryos. The embryos were allowed to develop until stages 28 to 30. The epidermis and the somites were dissected, or the embryos were left intact before protein and RNA extractions. (A) From top to bottom, Western blots using antibodies against Ptbp1, the V5 tag, and Pcna (loading control); results of one representative semiquantitative RT-PCR experiment using primers targeting eef1a1 and ptbp1 mRNAs, where the reverse primer targets the 3′ untranslated region of endogenous (endog.) ptbp1 mRNA (exon 15) and does not amplify the injected recombinant mRNA; and quantification of the ptbp1/eef1a1 mRNA ratios (mean ± SD from 3 independent experiments). (B) (Top) Schematic drawing of ptbp1 pre-mRNA. Exon 11 is either spliced or skipped. The arrows reveal the positions of the PCR primers (forward primer in exon 10 and reverse primer in exon 13). (Middle) Results of a representative semiquantitative RT-PCR. (Bottom) The percentage of ptbp1 mRNA excluding exon 11 (dark gray, ptbp1 mRNA without exon 11/total ptbp1 mRNA) and the total ptbp1/eef1a1 mRNA ratios (light gray) from 3 independent experiments (mean ± SD).
	FIG 5 Control of ptbp1 mRNA splicing pattern by Esrp1. (A and B) The indicated molecules were injected in both blastomeres of two-cell embryos. The embryos were allowed to develop until stage 15 (A) or stages 28 to 30 (B) before RNA extraction. The splicing pattern of ptbp1 mRNA was analyzed as described in the legend to Fig. 4B. (Top) Results of one representative experiment; (bottom) quantifications (mean ± SD from 3 independent experiments). (A) Injection of esrp1 MO and/or a recombinant esrp1-V5 RNA immune to the esrp1 MO. (B) Injection of morpholinos (60 ng control MO, 20 ng ptbp1 MO, 10, 20, or 40 ng esrp1 MO). (C) (Top) Schematic drawing of ptbp1 pre-mRNA. Usage of the proximal (prox.) 5′ splice site (ss) in intron 2 produces an mRNA with a short exon 2 (exon 2a). The arrows in exons 1 and 3 reveal the positions of the PCR primers. (Middle and bottom) Results of one representative experiment (middle) and quantifications (bottom; mean ± SD from 3 independent experiments), as in panel A. (D) (Top) Structure of the minigene, which encompasses the keratin promoter and the region of the ptbp1 gene between exons 10 and 12. The splicing pattern of the RNA transcribed from the minigene is assayed using primers flanking exon 11 (arrows). This assay is insensitive to the endogenous ptbp1 mRNA because both primers target vector (v) sequences and are therefore specific to the minigene. (Bottom) The minigene was injected with the indicated MO in one blastomere of two-cell embryos. The embryos were arrested at stages 28 to 30, before RNA extraction and analyses of splicing pattern. (Bottom) Quantification from 3 independent experiments (mean ± SD).
	FIG 6 Esrp1 binds to ptbp1 pre-mRNA. (A) An mRNA encoding a V5-tagged Esrp1 protein was injected in both blastomeres of two-cell embryos that were allowed to develop until stage 15. The V5-tagged protein was immunoprecipitated using anti-V5 antibodies or IgGs for mock immunoprecipitations. (Top) The input, the flowthrough (FT), and the immunoprecipitated fractions analyzed by Western blotting using antibodies against V5, Ptbp1, and Pcna. Equivalent amounts of the input, flowthrough, and eluates were loaded. (Bottom) Measurement of the amounts of ptbp1 pre-mRNA and mRNA in the immunoprecipitated fractions relative to the input amount using primers for the indicated intron (i) and exon (e) pairs. These immunoprecipitated fraction-to-input ratios (mean ± SD) result from 3 independent anti-V5 and mock immunoprecipitations. (B) (Left) Matrices were obtained by PCR amplification using combinations of forward and reverse primers, indicated by arrows. They were used for in vitro transcription to obtain 4 radiolabeled RNAs encompassing different fragments of intron 10, exon 11, and intron 11 of ptbp1 pre-mRNA. IP, immunoprecipitation. (Right) The in vitro transcripts were incubated in protein extracts made from embryos previously injected with esrp1-V5 mRNA (top) or ptbp1-V5 mRNA (middle) or left uninjected (bottom). After UV cross-linking and RNase treatment, the V5-tagged proteins were immunoprecipitated, electrophoresed, and revealed by autoradiography.
	FIG 7 Model for the control of Ptbp1 abundance. The ptbp1 pre-mRNA matures into two isoforms differing by the presence of exon 11. The isoform devoid of exon 11 (right) contains a premature stop codon and is targeted to rapid degradation by NMD. The isoform containing exon 11 (left) is translated into the Ptbp1 protein. Exon 11 is spliced with a low efficiency, and Esrp1 is required to achieve exon 11 inclusion and, thereby, Ptbp1 accumulation in the epidermis. Ptbp1 counteracts Esrp1 to favor exon 11 skipping, which provides the basis for the ptbp1 self-regulatory feedback loop. Rbm4 also reduces exon 11 inclusion in muscle, heart, and, potentially, other tissues. Additional unknown trans-acting factors X may stimulate exon 11 splicing or skipping.

Anquetil, Polypyrimidine tract binding protein prevents activity of an intronic regulatory element that promotes usage of a composite 3'-terminal exon. 2009, Pubmed, Xenbase

Anquetil, Polypyrimidine tract binding protein prevents activity of an intronic regulatory element that promotes usage of a composite 3'-terminal exon. 2009, Pubmed , Xenbase
Ayala, TDP-43 regulates its mRNA levels through a negative feedback loop. 2011, Pubmed
Carstens, An intronic splicing silencer causes skipping of the IIIb exon of fibroblast growth factor receptor 2 through involvement of polypyrimidine tract binding protein. 2000, Pubmed
Castelo-Branco, Polypyrimidine tract binding protein modulates efficiency of polyadenylation. 2004, Pubmed
Chou, Multisite RNA binding and release of polypyrimidine tract binding protein during the regulation of c-src neural-specific splicing. 2000, Pubmed
Dai, RNA-binding protein HuR autoregulates its expression by promoting alternative polyadenylation site usage. 2012, Pubmed
Dembowski, The CUGBP2 splicing factor regulates an ensemble of branchpoints from perimeter binding sites with implications for autoregulation. 2009, Pubmed
de Melo Neto, Autoregulation of poly(A)-binding protein synthesis in vitro. 1995, Pubmed
Dittmar, Genome-wide determination of a broad ESRP-regulated posttranscriptional network by high-throughput sequencing. 2012, Pubmed
Duriez, Alternative splicing of Xenopus alphafast-tropomyosin pre-mRNA during development: identification of determining sequences. 2000, Pubmed , Xenbase
Hamon, Polypyrimidine tract-binding protein is involved in vivo in repression of a composite internal/3' -terminal exon of the Xenopus alpha-tropomyosin Pre-mRNA. 2004, Pubmed , Xenbase
Hardy, Characterization of muscle and non muscle Xenopus laevis tropomyosin mRNAs transcribed from the same gene. Developmental and tissue-specific expression. 1991, Pubmed , Xenbase
Hardy, Two skeletal alpha-tropomyosin transcripts with distinct 3'UTR have different temporal and spatial patterns of expression in the striated muscle lineages of Xenopus laevis. 1999, Pubmed , Xenbase
Heyd, Getting under the skin of alternative splicing: identification of epithelial-specific splicing factors. 2009, Pubmed
Horiguchi, TGF-β drives epithelial-mesenchymal transition through δEF1-mediated downregulation of ESRP. 2012, Pubmed
Huang, Phosphorylation by SR kinases regulates the binding of PTB-associated splicing factor (PSF) to the pre-mRNA polypyrimidine tract. 2007, Pubmed
Huang, A molecular link between SR protein dephosphorylation and mRNA export. 2004, Pubmed
Izquierdo, Regulation of Fas alternative splicing by antagonistic effects of TIA-1 and PTB on exon definition. 2005, Pubmed
Jensen, Nova-1 regulates neuron-specific alternative splicing and is essential for neuronal viability. 2000, Pubmed
Jumaa, The splicing factor SRp20 modifies splicing of its own mRNA and ASF/SF2 antagonizes this regulation. 1997, Pubmed
Klein, Genetic and genomic tools for Xenopus research: The NIH Xenopus initiative. 2002, Pubmed , Xenbase
Le Guiner, TIA-1 and TIAR activate splicing of alternative exons with weak 5' splice sites followed by a U-rich stretch on their own pre-mRNAs. 2001, Pubmed
Le Sommer, PTB regulates the processing of a 3'-terminal exon by repressing both splicing and polyadenylation. 2005, Pubmed , Xenbase
Lin, RBM4 down-regulates PTB and antagonizes its activity in muscle cell-specific alternative splicing. 2011, Pubmed
Lin, Exon selection in alpha-tropomyosin mRNA is regulated by the antagonistic action of RBM4 and PTB. 2005, Pubmed
Llorian, Position-dependent alternative splicing activity revealed by global profiling of alternative splicing events regulated by PTB. 2010, Pubmed
Lu, Exo70 isoform switching upon epithelial-mesenchymal transition mediates cancer cell invasion. 2013, Pubmed
Makeyev, The MicroRNA miR-124 promotes neuronal differentiation by triggering brain-specific alternative pre-mRNA splicing. 2007, Pubmed
Manley, SR proteins and splicing control. 1996, Pubmed
Noiret, Expression analysis of the polypyrimidine tract binding protein (PTBP1) and its paralogs PTBP2 and PTBP3 during Xenopus tropicalis embryogenesis. 2012, Pubmed , Xenbase
Pan, Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing. 2008, Pubmed
Revil, During embryogenesis, esrp1 expression is restricted to a subset of epithelial cells and is associated with splicing of a number of developmentally important genes. 2013, Pubmed
Rossbach, Auto- and cross-regulation of the hnRNP L proteins by alternative splicing. 2009, Pubmed
Schmid, The splicing landscape is globally reprogrammed during male meiosis. 2013, Pubmed
Shapiro, An EMT-driven alternative splicing program occurs in human breast cancer and modulates cellular phenotype. 2011, Pubmed
Singh, Building specificity with nonspecific RNA-binding proteins. 2005, Pubmed
Spellman, Crossregulation and functional redundancy between the splicing regulator PTB and its paralogs nPTB and ROD1. 2007, Pubmed
Stoilov, Human tra2-beta1 autoregulates its protein concentration by influencing alternative splicing of its pre-mRNA. 2004, Pubmed
Sun, SF2/ASF autoregulation involves multiple layers of post-transcriptional and translational control. 2010, Pubmed
Tacke, Sequence-specific RNA binding by an SR protein requires RS domain phosphorylation: creation of an SRp40-specific splicing enhancer. 1997, Pubmed
Ueda, Epithelial splicing regulatory protein 1 is a favorable prognostic factor in pancreatic cancer that attenuates pancreatic metastases. 2014, Pubmed
Wagner, Characterization of the intronic splicing silencers flanking FGFR2 exon IIIb. 2005, Pubmed
Wagner, Polypyrimidine tract binding protein antagonizes exon definition. 2001, Pubmed
Wang, Alternative isoform regulation in human tissue transcriptomes. 2008, Pubmed
Warzecha, ESRP1 and ESRP2 are epithelial cell-type-specific regulators of FGFR2 splicing. 2009, Pubmed , Xenbase
Warzecha, An ESRP-regulated splicing programme is abrogated during the epithelial-mesenchymal transition. 2010, Pubmed
Wollerton, Autoregulation of polypyrimidine tract binding protein by alternative splicing leading to nonsense-mediated decay. 2004, Pubmed
Xue, Genome-wide analysis of PTB-RNA interactions reveals a strategy used by the general splicing repressor to modulate exon inclusion or skipping. 2009, Pubmed
Xue, Direct conversion of fibroblasts to neurons by reprogramming PTB-regulated microRNA circuits. 2013, Pubmed
Yae, Alternative splicing of CD44 mRNA by ESRP1 enhances lung colonization of metastatic cancer cell. 2012, Pubmed
Yap, Coordinated regulation of neuronal mRNA steady-state levels through developmentally controlled intron retention. 2012, Pubmed
Ye, A pathway involving HDAC5, cFLIP and caspases regulates expression of the splicing regulator polypyrimidine tract binding protein in the heart. 2013, Pubmed
Zheng, PSD-95 is post-transcriptionally repressed during early neural development by PTBP1 and PTBP2. 2012, Pubmed