A double reporter assay for detecting changes in the ratio of spliced and unspliced mRNA in mammalian cells.
Current methods for measuring the efficiency of splicing in mammalian cells rely on either direct analysis of the RNA, which does not lend itself to rapid assays, or on single reporter functions that are subject to numerous intrinsic variables. If two protein activities are encoded within a single reading frame but on separate exons, with an intervening sequence containing termination codons, then the expression of the second activity is dependent on removal of the intervening sequence by pre-mRNA splicing. Thus, the ratio of the activities encoded by exon 2 to exon 1 reflects the ratio of expression from spliced mRNA to the total expression of spliced and unspliced RNA. This provides a rapid and convenient assay for the effects on splicing efficiency of trans-acting factors or of alterations in the sequences of the intron and surrounding exon sequences.
Nasim
Md Talat
MT
Department of Biochemistry, University of Leicester, University Road, Leicester LE1 7RH, UK.
Chowdhury
H M
HM
Eperon
I C
IC
D000818:Animals; D002460:Cell Line; D017930:Genes, Reporter; D005821:Genetic Techniques; D006801:Humans; D008156:Luciferases / Q000032:analysis / Q000235:genetics; D008322:Mammals; D012326:RNA Splicing; D012333:RNA, Messenger / Q000032:analysis* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000032:analysis; D001616:beta-Galactosidase / Q000032:analysis / Q000235:genetics
D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D008156:Luciferases; D001616:beta-Galactosidase