Identification of the shortest splice variant of Dp71, together with five known variants, in glioblastoma cells
Identification of the shortest splice variant of Dp71, together with five known variants, in glioblastoma cells
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DOI：
10.1016/j.bbrc.2018.11.168
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发表时间：
2019-01-08
影响因子：
3.1
通讯作者：
Matsuo, Masafumi
中科院分区：
文献类型：
--
作者：
Rani, Abdul Qawee Mahyoob;Farea, Manal;Matsuo, Masafumi
背景：Ddystrophin Dp71mRNA是由DMD基因最末端的替代启动子产生的，该基因的突变会导致Duchenne肌营养不良症(DMD)。Dp71具有多种剪接变异体的特征。除了与DMD患者的认知障碍有关外，Dp71还可能在肿瘤的发生中发挥作用。本研究分析了最常见和最致命的脑恶性肿瘤--胶质母细胞瘤中Dp71的转录本。方法：采用逆转录-聚合酶链式反应(RT-PCR)检测U-251胶质母细胞瘤细胞系中Dp71基因的表达。结果：RT-PCR扩增Dp71转录本5‘端扩增出预期大小的产物，提示Dp71启动子在胶质母细胞瘤中转录。扩增从外显子G1到DMD外显子79的全长Dp71，得到预期大小的产物，以及一条微弱的、较小的条带。对17个克隆进行测序，发现了6个不同的剪接变体，其中只有一个克隆的全长Dp71包含所有18个外显子。10个克隆缺失外显子78(Dp71b)，提示Dp71b是胶质母细胞瘤中Dp71的主要类型。另外，3个克隆缺失外显子71和78(Dp71ab)，1个克隆缺失外显子71、73和78(Dp71ab Delta 73)，1个克隆缺失外显子71-74和78(Dp71 BC)，1个克隆缺失外显子68-76和78(Dp71b Delta 68-76)。这个新的转录本是最短的Dp71变异体，在外显子77预测有一个终止密码子，预测产生一个24.8 kDa的蛋白质，由216个氨基酸组成，其中包括来自外显子77的15个氨基酸。结论：在胶质母细胞瘤细胞中发现了6种Dp71剪接变异体，其中Dp71b含量最高。外显子78的缺失是胶质母细胞瘤的一个明显的默认剪接途径，在17个克隆中有16个被观察到。胶质母细胞瘤细胞含有迄今发现的最短的Dp71转录本(Dp71b Delta 68-76)，具有独特的C-末端氨基酸序列。这些发现提示有必要评估Dp71变异体在胶质母细胞瘤中的功能。(C)2018年由Elsevier Inc.出版。
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 (Dp71 ab Delta 73), one clone lacked exons 71-74 and 78 (Dp71 bc), and one clone lacked exons 68-76 and 78 (Dp71b Delta 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 Delta 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. (C) 2018 Published by Elsevier Inc.