The CD44 alternative v9 exon contains a splicing enhancer responsive to the SR proteins 9G8, ASF/SF2, and SRp20.
The CD44 gene alternative exons v8, v9, and v10 are frequently spliced as a block by epithelial cells. By transfecting minigenes containing only one of these alternative exons, we show that splicing of each of them is under cell type-specific control. By using minigenes carrying short block mutations within exons v8 and v9, we detected a candidate exon splicing enhancer in each of these exons. These candidates activated splicing in vitro of a heterologous transcript and are thus true exon splicing enhancers. We analyzed further a v9 exon splicing enhancer covering approximately 30 nucleotides. This enhancer can be UV cross-linked to SR proteins of 35 and 20 kDa in HeLa nuclear extract. By using individual recombinant SR proteins for UV cross-linking in S100 extract, these proteins were identified as 9G8, ASF/SF2, and SRp20. S100 complementation studies using recombinant 9G8, ASF/SF2, and SRp20 showed that all three proteins can activate splicing in vitro of a heterologous exon containing the v9 enhancer; the strongest activation was obtained with 9G8. Progressive truncation of the 30-nucleotide enhancer leads to a progressive decrease in splicing activation. We propose that 9G8, ASF/SF2, SRp20, and possibly other non-SR proteins cooperate in vivo to activate v9 exon splicing.
Galiana-Arnoux
Delphine
D
INSERM U463, Institut de Biologie-CHR, 9 Quai Moncousu, 44093 Nantes Cedex 1, France.
Lejeune
Fabrice
F
Gesnel
Marie-Claude
MC
Stevenin
James
J
Breathnach
Richard
R
Del Gatto-Konczak
Fabienne
F
D017398:Alternative Splicing; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D004847:Epithelial Cells / Q000378:metabolism; D005091:Exons; D005816:Genetic Complementation Test; D006367:HeLa Cells; D006801:Humans; D018960:Hyaluronan Receptors / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D007438:Introns; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism*; D029543:Nucleocytoplasmic Transport Proteins / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000068103:Serine-Arginine Splicing Factors; D014162:Transfection; D014466:Ultraviolet Rays
D018960:Hyaluronan Receptors; D009687:Nuclear Proteins; D029543:Nucleocytoplasmic Transport Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; C491388:SRSF3 protein, human; D000068103:Serine-Arginine Splicing Factors