Polypyrimidine tract-binding protein is involved in vivo in repression of a composite internal/3' -terminal exon of the Xenopus alpha-tropomyosin Pre-mRNA.
The Xenopus alpha(fast)-tropomyosin gene contains, at its 3' -end, a composite internal/3' -terminal exon (exon 9A9'), which is subjected to three different patterns of splicing according to the cell type. Exon 9A9' is included as a terminal exon in the myotome and as an internal exon in adult striated muscles, whereas it is skipped in nonmuscle cells. We have developed an in vivo model based on transient expression of minigenes encompassing the regulated exon 9A9' in Xenopus oocytes and embryos. We first show that the different alpha-tropomyosin minigenes recapitulate the splicing pattern of the endogenous gene and constitute valuable tools to seek regulatory sequences involved in exon 9A9' usage. A mutational analysis led to the identification of an intronic element that is involved in the repression of exon 9A9' in nonmuscle cells. This element harbors four polypyrimidine track-binding protein (PTB) binding sites that are essential for the repression of exon 9A9'. We show using UV cross-linking and immunoprecipitation experiments that Xenopus PTB (XPTB) interacts with these PTB binding sites. Finally, we show that depletion of endogenous XPTB in Xenopus embryos using a morpholinobased translational inhibition strategy resulted in exon 9A9' inclusion in embryonic epidermal cells. These results demonstrate that XPTB is required in vivo to repress the terminal exon 9A9' and suggest that PTB could be a major actor in the repression of regulated 3' -terminal exon.
Hamon
Sandra
S
UMR 6061 CNRS, Université de Rennes 1, Faculté de Médecine, 35043 Rennes cedex, France.
Le Sommer
Caroline
C
Mereau
Agnès
A
Allo
Marie-Rose
MR
Hardy
Serge
S
D000199:Actins / Q000378:metabolism; D017398:Alternative Splicing; D020816:Amino Acid Motifs; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D003432:Cross-Linking Reagents / Q000494:pharmacology; D018076:DNA, Complementary / Q000378:metabolism; D000078404:Epidermal Cells; D005091:Exons; D005260:Female; D020868:Gene Silencing; D007438:Introns; D007633:Keratins / Q000378:metabolism; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism; D010053:Ovary / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D038941:Polypyrimidine Tract-Binding Protein / Q000502:physiology*; D011233:Precipitin Tests; D014176:Protein Biosynthesis; D012313:RNA; D012322:RNA Precursors / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014335:Tropomyosin / Q000378:metabolism*; D014466:Ultraviolet Rays; D014982:Xenopus laevis
D000199:Actins; D003432:Cross-Linking Reagents; D018076:DNA, Complementary; D012322:RNA Precursors; D014335:Tropomyosin; D038941:Polypyrimidine Tract-Binding Protein; D012313:RNA; D007633:Keratins