Department of Pathophysiology, Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima, 770-8503, Japan.
Student Lab, Tokushima University Faculty of Medicine, Tokushima, Japan.
Sci Rep. 2019 Jul 16;9(1):10276. doi: 10.1038/s41598-019-46659-x.
The human TRA2B gene consists of 10 exons and 9 introns and produces 5 splice isoforms (TRA2β1 to TRA2β5). TRA2B exon 2 encodes multiple premature termination codons. TRA2β1 lacks exon 2 and is translated into a functional transformer 2β (Tra2β) protein, whereas TRA2β4 contains 10 exons and works as a functional RNA. Overexpressed Tra2β and ectopic expression of TRA2β4 may be oncogenic. We found that heterogeneous nuclear ribonucleoprotein (hnRNP)A1 and hnRNPU interacted with TRA2β4 exon 2. Minigene assays revealed that hnRNPA1 facilitated inclusion of exon 2, whereas hnRNPU promoted its skipping. However, knockdown of hnRNPA1 or hnRNPU reduced both TRA2β1 and TRA2β4 levels, and overexpression of these hnRNPs increased levels of both isoforms, suggesting that hnRNPA1 and hnRNPU mainly regulate the transcription of TRA2B. In fact, hnRNPA1 and hnRNPU positively regulated the promoter activity of TRA2B. Circular dichroism analyses, electrophoretic mobility shift assays and chromatin immunoprecipitation assays demonstrated the presence of G-quadruplex (G4) formation in the promoter of TRA2B. Formation of G4 suppressed TRA2B transcription, whereas hnRNPA1, but not hnRNPU, interacted with the G4 to facilitate transcription. Our results suggest that hnRNPA1 may modulate TRA2B transcription through its regulation of G4 formation in its promoter in colon cancer cells.
人类 TRA2B 基因由 10 个外显子和 9 个内含子组成，产生 5 种剪接异构体（TRA2β1 至 TRA2β5）。TRA2B 外显子 2 编码多个提前终止密码子。TRA2β1 缺失外显子 2 并被翻译为功能性转录因子 2β（Tra2β）蛋白，而 TRA2β4 包含 10 个外显子并作为功能性 RNA 发挥作用。过表达的 Tra2β 和 TRA2β4 的异位表达可能具有致癌性。我们发现异质核核糖核蛋白（hnRNP）A1 和 hnRNPU 与 TRA2β4 外显子 2 相互作用。迷你基因试验表明，hnRNPA1 促进外显子 2 的包含，而 hnRNPU 促进其跳过。然而，hnRNPA1 或 hnRNPU 的敲低均降低了 TRA2β1 和 TRA2β4 的水平，并且这些 hnRNPs 的过表达增加了两种异构体的水平，表明 hnRNPA1 和 hnRNPU 主要调节 TRA2B 的转录。事实上，hnRNPA1 和 hnRNPU 正向调节 TRA2B 的启动子活性。圆二色性分析、电泳迁移率变动分析和染色质免疫沉淀分析表明 TRA2B 启动子中存在 G-四链体（G4）形成。G4 的形成抑制 TRA2B 转录，而 hnRNPA1 而非 hnRNPU 与 G4 相互作用以促进转录。我们的结果表明，hnRNPA1 可能通过调节其启动子中的 G4 形成来调节结肠癌细胞中 TRA2B 的转录。
