An exonic splicing enhancer offsets the atypical GU-rich 3' splice site of human apolipoprotein A-II exon 3.
Human apolipoprotein A-II (apoA-II) intron 2/exon 3 junction shows a peculiar tract of alternating pyrimidines and purines (GU tract) that makes the acceptor site deviate significantly from the consensus. However, apoA-II exon 3 is constitutively included in mRNA. We have studied this unusual exon definition by creating a construct with the genomic fragment encompassing the whole gene from apoA-II and its regulatory regions. Transient transfections in Hep3B cells have shown that deletion or replacement of the GU repeats at the 3' splice site resulted in a decrease of apoA-II exon 3 inclusion, indicating a possible role of the GU tract in splicing. However, a 3' splice site composed of the GU tract in heterologous context, such as the extra domain A of human fibronectin or cystic fibrosis transmembrane conductance regulator exon 9, resulted in total skipping of the exons. Next, we identified the exonic cis-acting elements that may affect the splicing efficiency of apoA-II exon 3 and found that the region spanning from nucleotide 87 to 113 of human apoA-II exon 3 is essential for its inclusion in the mRNA. Overlapping deletions and point mutations (between nucleotides 91 and 102) precisely defined an exonic splicing enhancer (ESEwt). UV cross-linking assays followed by immunoprecipitation with anti-SR protein monoclonal antibodies showed that ESEwt, but not mutated ESE RNA, was able to bind both alternative splicing factor/splicing factor 2 and SC35. Furthermore, overexpression of both splicing factors enhanced exon 3 inclusion. These results show that this protein-ESE interaction is able to promote the incorporation of exon 3 in mRNA and suggest that they can rescue the splicing despite the noncanonical 3' splice site.
Arrisi-Mercado
Pablo
P
International Centre for Genetic Engineering and Biotechnology, Padriciano 99, I-34012 Trieste, Italy.
Romano
Maurizio
M
Muro
Andres F
AF
Baralle
Francisco E
FE
D016633:Apolipoprotein A-II / Q000235:genetics*; D001120:Arginine; D001483:Base Sequence; D006528:Carcinoma, Hepatocellular; D045744:Cell Line, Tumor; D002874:Chromosome Mapping; D024202:Electrophoretic Mobility Shift Assay; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D005091:Exons / Q000235:genetics; D005353:Fibronectins / Q000737:chemistry / Q000235:genetics; D005816:Genetic Complementation Test; D006147:Guanine; D006801:Humans; D007438:Introns / Q000235:genetics; D008113:Liver Neoplasms; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009687:Nuclear Proteins / Q000378:metabolism; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D022821:RNA Splice Sites / Q000235:genetics*; D012326:RNA Splicing / Q000235:genetics*; D016601:RNA-Binding Proteins; D012694:Serine; D000068103:Serine-Arginine Splicing Factors
D016633:Apolipoprotein A-II; D005353:Fibronectins; D009687:Nuclear Proteins; D022821:RNA Splice Sites; D016601:RNA-Binding Proteins; D000068103:Serine-Arginine Splicing Factors; D012694:Serine; D006147:Guanine; D001120:Arginine