Identification of the shortest splice variant of Dp71, together with five known variants, in glioblastoma cells

Abstract

Background: Dystrophin Dp71 mRNA is produced from the most distal alternative promoter of the DMD gene, mutations in which cause Duchenne muscular dystrophy (DMD). Dp71 is characterized by a wide variety of splice variants. In addition to being associated with cognitive disturbance in patients with DMD, Dp71 may also play a role in tumorigenesis. This study analyzed Dp71 transcripts in glioblastoma, the most common and most lethal type of cerebral malignancy.

Methods: Dp71 mRNA in the U-251 glioblastoma cell line was analyzed by reverse-transcription polymerase chain reaction (RT-PCR). The amplified products were subcloned and sequenced.

Results: RT-PCR amplification of the 5' end of the Dp71 transcript yielded a product of expected size, indicating transcription from the Dp71 promoter in glioblastoma. Amplification of full-length Dp71, from exon G1 to DMD exon 79, yielded a product of expected size, as well as a faint, smaller sized band. Sequencing of 17 clones revealed six different alternatively spliced variants, with only one clone being of full-length Dp71 containing all 18 exons. Ten clones lacked exon 78 (Dp71b), indicating that Dp71b was a major type of Dp71 in glioblastoma. In addition, three clones lacked both exons 71 and 78 (Dp71ab), one clone lacked exons 71, 73 and 78 (Dp71ab △73), one clone lacked exons 71-74 and 78 (Dp71bc), and one clone lacked exons 68-76 and 78 (Dp71b△68-76). This novel transcript was the shortest Dp71 variant, with a predicted stop codon in exon 77 and was predicted to produce a 24.8 kDa protein, consisting of 216 amino acids including 15 amino acids from exon 77. This novel product was classified as Dp71g because of its unique C-terminal amino acid sequence.

Conclusions: Six splice variants of Dp71 were identified in glioblastoma cells, with Dp71b being the most abundant. Deletion of exon 78 was an apparent default splicing pathway in glioblastoma, being observed in 16 of 17 clones. Glioblastoma cells contained the shortest Dp71 transcript (Dp71b△68-76) identified to date, with a unique C-terminal amino acid sequence. These findings suggest the need to assess the function of Dp71 variants in glioblastoma.

Keywords: Dp71; Dystrophin; Glioblastoma; Splice variant; U251 cells.