Molecular characterization of the 5′-UTR of retinal dystrophin reveals a cryptic intron that regulates translational activity

PURPOSE: Mutations in the dystrophin (DMD) gene cause Duchenne or Becker muscular dystrophy (DMD/BMD). DMD contains a retina-specific promoter in intron 29. The short R-dystrophin transcript from this promoter has a retina-specific exon 1 (R1) joined to exon 30 of the DMD gene. It has been claimed that this is responsible for the ophthalmological problems observed in DMD/BMD. This research characterizes the structure of the 5′-untranslated region (5′-UTR) of human R-dystrophin. METHODS: The 5′-UTR of the human R-dystrophin transcript was amplified from human retina and 20 other human tissue RNAs by reverse transcription polymerase chain reaction (RT–PCR). Amplified products were identified by sequencing. The translational activities of transcripts bearing differing 5′-UTRs were measured using a dual luciferase assay system. RESULTS: RT–PCR amplification of the R-dystrophin transcript from the retina using a conventional primer set revealed one product comprising exon R1 and exons 30 to 32 (R-dys α). In contrast, three amplified products were obtained when a forward primer at the far 5′-end of exon R1 was employed for RT–PCR. R-dys α, and a shorter form in which 98 bp was deleted from exon R1 (R-dys β), were the two major products. A minor, short form was also identified, in which 143 bp was deleted from exon R1 (R-dys γ). The two primary retinal products (R-dys α and β) encoded an identical open reading frame. The 98 bp deleted in R-dys β was identified as a cryptic intron that was evolutionarily acquired in higher mammals. The shorter R-dys β was expressed in several tissues with a wide range in expression level, while R-dys α was retina specific. The 5′-UTRs of R-dys α and β were examined for translational activity using a dual luciferase assay system. Unexpectedly, the 5′-UTR of R-dys β showed lower translational activity than that of R-dys α. This lower activity was presumed to be due to the removal of internal ribosome entry sites by activation of cryptic intron splicing. CONCLUSIONS: An evolutionarily-acquired cryptic intron was identified in the 5′-UTR of the human R-dystrophin transcript. The two abundant R-dystrophin transcripts in the retina showed different translational activities in vitro owing to their differential splicing of the cryptic intron. This evolutionarily-acquired alternative splicing may act as a molecular switch that regulates translation of the R-dystrophin transcript.