Figure 3

Minigene analysis of the effect of the c.659-131 T > G mutation on GPR143 pre-mRNA splicing.

(a) Diagrams of intron 5 and flanking exons 5 and 6 where the deep cryptic mutation c.659-131 T > G is located (upper) and the pET01-INT5 construct (lower). In the pET01-INT5 construct, the intron containing the multiple cloning site (MCS) is framed by the 5’-donor and 3’-acceptor splice sites of the preproinsulin exon (http://www.mobitec.com/cms/products/bio/04_vector_sys/exontrap.html). The expression of this vector sequence is driven by the promoter present in the long terminal repeat (LTR) of Rous Sarcoma Virus followed by a short stretch of a eukaryotic gene (phosphatase). The mutation detected in GPR143 intron 5 (arrowhead) creates a cryptic donor splice site and generates a pseudoexon (Ψ5b) of 41 bp in the mRNA. Primers used for RT-PCR experiments are indicated by the arrows. The length of each fragment is also indicated. (b) RT-PCR analysis of the correct 127-bp amplicon when HEK293 and HCT116 cells were transfected with the wild-type (WT) pET01-INT5-WT minigene. The 168-bp transcript, including cryptic exon (Ψ5b), was mainly detected in HEK293 and HCT116 cells transfected with the mutated minigene (pET01-INT5-MUT, MUT) as schematically indicated to the right of the gel. No product was detected in cells transfected with the empty pET01 vector (Mock).(c) Nucleotide and amino acid sequences. An arrow denotes an exon–exon boundary. Sequence analysis identified a 41-bp pseudoexon between GPR143 exons 5 and 6 of the affected individuals with the c.659-131 T > G mutation. This pseudoexon introduces a premature termination codon after 10 amino acid residues. Sequence analysis of an unaffected individual with the wild-type allele indicated normal splicing of exons 5 and 6.