Cellular Expression and siRNA-Mediated Interference of Rhodopsincis-Acting Splicing Mutants Associated with Autosomal Dominant Retinitis Pigmentosa

Imma Hernan, María José Gamundi, Ester Planas, Emma Borràs, Miquel Maseras, Miguel Carballo
2011 Investigative Ophthalmology and Visual Science  
PURPOSE. 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.531-2AϾG, c.936ϩ1GϾT,
more » ... 936ϩ1GϾT, c.937-1GϾT and c.745GϾT 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.531-2AϾG and c.937-1GϾT, induce cryptic splicing. In contrast, the c.936ϩ1GϾT mutation, which causes arRP, results in exon skipping. Although the c.531-2AϾG and c.745GϾT 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. (Invest Ophthalmol Vis Sci.
doi:10.1167/iovs.10-6933 pmid:21357407 fatcat:xsiqnwq62jhmnmam7rm6equzwe