Kubokawa Ikuko, Takeshima Yasuhiro, Ota Mitsunori, Enomoto Masahiro, Okizuka Yo, Mori Takeshi, Nishimura Noriyuki, Awano Hiroyuki, Yagi Mariko, Matsuo Masafumi
Department of Pediatrics, Graduate School of Medicine, Kobe University, Kobe, Japan.
Mol Vis. 2010 Dec 7;16:2590-7.
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.
肌营养不良蛋白（DMD）基因突变会导致杜氏或贝克型肌营养不良症（DMD/BMD）。DMD基因的第29号内含子含有一个视网膜特异性启动子。来自该启动子的短R-肌营养不良蛋白转录本有一个视网膜特异性外显子1（R1）与DMD基因的外显子30相连。据称，这是导致DMD/BMD患者出现眼科问题的原因。本研究对人R-肌营养不良蛋白5'-非翻译区（5'-UTR）的结构进行了表征。
通过逆转录聚合酶链反应（RT-PCR）从人视网膜和其他20种人体组织RNA中扩增人R-肌营养不良蛋白转录本的5'-UTR。通过测序鉴定扩增产物。使用双荧光素酶检测系统测量携带不同5'-UTR的转录本的翻译活性。
使用常规引物对从视网膜进行R-肌营养不良蛋白转录本的RT-PCR扩增，得到一个包含外显子R1和外显子30至32的产物（R-dysα）。相比之下，当使用位于外显子R1 5'-末端远端的正向引物进行RT-PCR时，获得了三种扩增产物。R-dysα和一种从外显子R1缺失98 bp的较短形式（R-dysβ）是两种主要产物。还鉴定出一种次要的短形式，其中外显子R1缺失了143 bp（R-dysγ）。两种主要的视网膜产物（R-dysα和β）编码相同的开放阅读框。R-dysβ中缺失的98 bp被鉴定为一个在高等哺乳动物中进化获得的隐蔽内含子。较短的R-dysβ在几种组织中表达，表达水平差异很大，而R-dysα是视网膜特异性的。使用双荧光素酶检测系统检测R-dysα和β的5'-UTR的翻译活性。出乎意料的是，R-dysβ的5'-UTR显示出比R-dysα更低的翻译活性。这种较低的活性被认为是由于隐蔽内含子剪接的激活导致内部核糖体进入位点的去除。
在人R-肌营养不良蛋白转录本的5'-UTR中鉴定出一个进化获得的隐蔽内含子。视网膜中两种丰富的R-肌营养不良蛋白转录本由于其对隐蔽内含子的不同剪接，在体外显示出不同的翻译活性。这种进化获得的可变剪接可能作为一种分子开关来调节R-肌营养不良蛋白转录本 的翻译。
