DEFECTIVE RNA SPLICING RESULTING FROM A MUTATION THE CYCLIC GUANOSINEMONOPHOSPHATE-PHOSPHODIESTERASE BETA-SUBUNIT GENE

PURPOSE. The authors have previously reported a 3' splice site mutatio n in intron 2 of the rod cyclic guanosine monophosphate-phosphodiester ase (cGMP)beta-subunit (beta-PDE) gene in a patient with compound hete rozygous autosomal recessive retinitis pigmentosa. The purpose of this study is to determine whether this mutation interferes with RNA splic ing, what products it generates, and whether the resultant mRNA is abl e to support the synthesis of the native protein. METHODS. TWO express ion constructs were prepared by subcloning genomic DNA fragments (one from the control subject DNA and the other from the patient's DNA) to the pCIS2 expression vector. Recombinant plasmid DNA was introduced in to 293 human embryonic kidney cells using the calcium phosphate-mediat ed transfection procedure. Northern blot hybridization, reverse transc ription-polymerase chain reaction, and sequencing were used for RNA an alysis. RESULTS. Four major products were present in the RNA pool isol ated from cells transfected with the expression construct containing t he splice site mutation. One of the transcripts resulted from the acti vation of a cryptic 3' splice site located in exon 3, 12 nucleotides d ownstream of the mutated site. The second fragment was longer than the correctly spliced mRNA by approximately 1 kb and contained unspliced intron 2. Two other high molecular weight products corresponded to int ermediate lariats. CONCLUSIONS. An acceptor splice site mutation in in tron 2 of the beta-PDE gene leads to the accumulation of pre-mRNA and intermediate lariats and a 12-nucleotide shorter beta-PDE transcript p roduced by the use of a cryptic splice site located in exon 3. In the normal beta-PDE mRNA, these 12 nucleotides code for ValPheLeuLys. Thes e amino acids are highly conserved in the putative noncatalytic cGMP-b inding domain I of beta-PDE from several species and, probably, are im portant for the correct folding and function of the protein.