title: Cellular expression and siRNA-mediated interference of rhodopsin cis-acting splicing mutants associated with autosomal dominant retinitis pigmentosa.
abstract: OBJECTIVE
  To investigate the cellular expression of cis-acting splicing mutations in the rhodopsin gene (RHO) that lead to autosomal dominant or recessive retinitis pigmentosa (adRP/arRP) and the role of nonsense-mediated mRNA decay (NMD) in its pathogenic mechanism. To design a potential therapeutic RNAi-based suppression strategy for cis-acting adRP splicing mutants.
  
  
  METHODS
  Cells were transfected with genomic constructs encoding the human wild-type (WT) and c.531-2A>G, c.936+1G>T, c.937-1G>T and c.745G>T RHO mutants. Total RNA was quantified by RT-PCR and protein was analyzed by immunocytochemistry. Three small interfering (si)RNAs directed against adRP mutant transcripts were designed and assayed in COS7 cells.
  
  
  RESULTS
  The RHO cis-acting splicing mutations causing adRP, c.531-2A>G and c.937-1G>T, induce cryptic splicing. In contrast, the c.936+1G>T mutation, which causes arRP, results in exon skipping. Although the c.531-2A>G and c.745G>T RHO sequence predicted a premature termination codon (PTC) that should be a target for NMD, these mutant proteins were detected in transfected cells. The siRNAs designed to interfere with adRP mutants silenced the corresponding mRNA with varying efficiency.
  
  
  CONCLUSIONS
  Although two RHO mutations that cause different RP phenotypes were the target for the NMD mechanism, a fraction of mutant RNA transcript may circumvent the NMD mechanism and be translated into protein. Thus, different levels of mutant protein may be necessary to trigger the RP phenotype. The findings demonstrate the potential use of siRNA to interfere with cis-acting splicing RHO transcripts. However, limitations in the mutation sequence and incomplete mutant transcript elimination should be considered in a therapeutic approach for adRP.
authors.affiliation: Molecular Genetics Laboratory, Hospital de Terrassa, Terrassa, Spain.
authors.forename: Imma
authors.initials: I
authors.lastname: Hernan
authors.forename: María José
authors.initials: MJ
authors.lastname: Gamundi
authors.forename: Ester
authors.initials: E
authors.lastname: Planas
authors.forename: Emma
authors.initials: E
authors.lastname: Borràs
authors.forename: Miquel
authors.initials: M
authors.lastname: Maseras
authors.forename: Miguel
authors.initials: M
authors.lastname: Carballo
mesh_terms: D017398:Alternative Splicing / Q000235:genetics; D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D005799:Genes, Dominant; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics; D009154:Mutation / Q000235:genetics*; D034622:RNA Interference; D012326:RNA Splicing / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D012174:Retinitis Pigmentosa / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012243:Rhodopsin / Q000235:genetics*; D014162:Transfection
chemical_list: D050505:Mutant Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D012243:Rhodopsin