Dai Guimin, Cheng Jiawen, Liu Weiran, Yin Xueli, Zhang Yuanyuan
School of Life Science, Anhui Medical University, Hefei 230032, China.
School of Basic Medical Sciences, Anhui Medical University, Hefei 230032, China.
Int J Mol Sci. 2025 Jan 29;26(3):1163. doi: 10.3390/ijms26031163.
This study developed an artificial chimeric intron module with an RNA riboswitch and TetR aptamer that were integrated into essential gene exons. Doxycycline can modulate Pre-mRNA alternative splicing, modify the exon reading frame, and dynamically regulate gene expression. By shifting the aptamer 2 base pair within the switch, we unexpectedly obtained the "on-switch" CTM and "off-switch" C2ITetR>4A, which possess thoroughly contrasting regulatory functions. The CTM module can conditionally induce tumor cell apoptosis and regulate genes reversibly and sustainably following doxycycline induction. We integrated the C2ITetR>4A/CTM switches with the L7Ae/k-turn module to create an intron-spliced double-switched RNA cascade system. The system can both activate and inhibit the splicing mechanism utilizing the same ligand to minimize crosstalk among aptamer switching elements, control target gene leakage, and enhance the dynamic range of gene expression. We analyzed numerous factors affecting Pre-mRNA splicing to identify the optimal equilibrium point for switch regulation. This will enable precise predictions of dynamic regulatory efficiency and the rational design of genetic modules, thereby providing a valuable instrument for mammalian synthetic biology.
本研究开发了一种人工嵌合内含子模块，其带有整合到必需基因外显子中的RNA核糖开关和TetR适体。强力霉素可调节前体mRNA可变剪接，改变外显子阅读框，并动态调控基因表达。通过在开关内将适体移动2个碱基对，我们意外获得了具有完全相反调控功能的“开启开关”CTM和“关闭开关”C2ITetR>4A。CTM模块可在强力霉素诱导后有条件地诱导肿瘤细胞凋亡，并可逆且可持续地调控基因。我们将C2ITetR>4A/CTM开关与L7Ae/k-turn模块整合，创建了一个内含子剪接双开关RNA级联系统。该系统可利用相同配体激活和抑制剪接机制，以最小化适体开关元件之间的串扰，控制靶基因泄漏，并增强基因表达的动态范围。我们分析了影响前体mRNA剪接的多种因素，以确定开关调控的最佳平衡点。这将能够精确预测动态调控效率并合理设计遗传模块，从而为哺乳动物合成生物学提供有价值的工具。
