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.