Cellular and viral splicing factors can modify the splicing pattern of CFTR transcripts carrying splicing mutations.
Variable levels of aberrantly spliced cystic fibrosis transmembrane conductance regulator (CFTR ) transcripts were suggested to correlate with variable cystic fibrosis (CF) severity. We studied the effect of the cellular splicing factors, hnRNP A1 and ASF/SF2, and their adenoviral analogues, E4-ORF6 and E4-ORF3, that promote exon skipping and/or exon inclusion, on the splicing pattern of the CFTR mutation 3849+10kb C-->T and the 5T allele. These mutations can lead to cryptic exon inclusion and exon skipping, respectively. Overexpression of the cellular factors promoted exon skipping of pre-mRNA transcribed from minigenes carrying the mutation (p5T or p3849M). This led to a substantial decrease in the level of correctly spliced mRNA transcribed from p5T and generated correctly spliced mRNA transcribed from p3849M that was not found without overexpression of the factors. The viral factor, E4-ORF3, promoted exon inclusion and led to a substantial increase of the correctly spliced mRNA transcribed from the p5T. The factor, E4-ORF6, activated exon skipping and generated correctly spliced mRNA transcribed from p3849M. Thus, overexpression of alternative splicing factors can modulate the splicing pattern of CFTR alleles carrying splicing mutations. These results are important for understanding the mechanism underlying phenotypic variability in CF and other genetic diseases.
Department of Genetics, Life Sciences Institute, The Hebrew University, Jerusalem 91904, Israel.
M
M
Nissim-Rafinia
O
O
Chiba-Falek
G
G
Sharon
A
A
Boss
B
B
Kerem
D016475:3T3 Cells; D017504:Adenovirus E4 Proteins / Q000235:genetics / Q000378:metabolism*; D017398:Alternative Splicing; D000818:Animals; D019556:COS Cells; D003550:Cystic Fibrosis / Q000235:genetics*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics*; D019073:HT29 Cells; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D006801:Humans; D051379:Mice; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D011071:Poly T; D012332:RNA, Heterogeneous Nuclear; D012333:RNA, Messenger; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000235:genetics / Q000378:metabolism*; D000068103:Serine-Arginine Splicing Factors; D014407:Tumor Cells, Cultured
D017504:Adenovirus E4 Proteins; C505032:CFTR protein, human; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D009687:Nuclear Proteins; D012332:RNA, Heterogeneous Nuclear; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000068103:Serine-Arginine Splicing Factors; D011071:Poly T