title: Defective RNA splicing resulting from a mutation in the cyclic guanosine monophosphate-phosphodiesterase beta-subunit gene.
abstract: OBJECTIVE
  The authors have previously reported a 3' splice site mutation in intron 2 of the rod cyclic guanosine monophosphate-phosphodiesterase (cGMP) beta-subunit (beta-PDE) gene in a patient with compound heterozygous autosomal recessive retinitis pigmentosa. The purpose of this study is to determine whether this mutation interferes with RNA splicing, what products it generates, and whether the resultant mRNA is able to support the synthesis of the native protein.
  
  
  METHODS
  Two expression 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 into 293 human embryonic kidney cells using the calcium phosphate-mediated transfection procedure. Northern blot hybridization, reverse transcription-polymerase chain reaction, and sequencing were used for RNA analysis.
  
  
  RESULTS
  Four major products were present in the RNA pool isolated from cells transfected with the expression construct containing the splice site mutation. One of the transcripts resulted from the activation of a cryptic 3' splice site located in exon 3, 12 nucleotides downstream 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 intermediate lariats.
  
  
  CONCLUSIONS
  An acceptor splice site mutation in intron 2 of the beta-PDE gene leads to the accumulation of pre-mRNA and intermediate lariats and a 12-nucleotide shorter beta-PDE transcript produced by the use of a cryptic splice site located in exon 3. In the normal beta-PDE mRNA, these 12 nucleotides code for ValPheLeuLys. These amino acids are highly conserved in the putative noncatalytic cGMP-binding domain I of beta-PDE from several species and, probably, are important for the correct folding and function of the protein.
authors.affiliation: Jules Stein Eye Institute, University of California, Los Angeles School of Medicine 90095, USA.
authors.forename: N I
authors.initials: NI
authors.lastname: Piriev
authors.forename: J M
authors.initials: JM
authors.lastname: Shih
authors.forename: D B
authors.initials: DB
authors.lastname: Farber
mesh_terms: D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000235:genetics* / Q000378:metabolism; D017398:Alternative Splicing / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D002460:Cell Line; D002478:Cells, Cultured; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6; D004247:DNA / Q000032:analysis; D017931:DNA Primers / Q000737:chemistry; D006801:Humans; D007668:Kidney / Q000201:enzymology; D008969:Molecular Sequence Data; D010727:Phosphoric Diester Hydrolases; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D012313:RNA / Q000032:analysis; D012326:RNA Splicing; D012174:Retinitis Pigmentosa / Q000201:enzymology / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection
chemical_list: D017931:DNA Primers; D012313:RNA; D004247:DNA; D010727:Phosphoric Diester Hydrolases; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6; C517431:PDE6B protein, human