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

• Published in: Molecular vision Vol. 16; pp. 2590 - 2597
• Main Authors: Kubokawa, Ikuko, Takeshima, Yasuhiro, Ota, Mitsunori, Enomoto, Masahiro, Okizuka, Yo, Mori, Takeshi, Nishimura, Noriyuki, Awano, Hiroyuki, Yagi, Mariko, Matsuo, Masafumi
• Format: Journal Article
• Language: English
• Published: United States Molecular Vision 07.12.2010
• Subjects: 5' Untranslated Regions - genetics; Alternative Splicing - genetics; Base Sequence; Dystrophin - genetics; Dystrophin - metabolism; Exons - genetics; Gene Expression Profiling; Genome, Human - genetics; HEK293 Cells; Humans; Introns - genetics; Molecular Sequence Data; Protein Biosynthesis - genetics; Retina - metabolism; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger - genetics; RNA, Messenger - metabolism
• ISSN: 1090-0535; 1090-0535

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. 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. 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. 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.