Chen Lei, Gao Xia, Liu Xiangshen, Zhu Ye, Wang Dong
Department of Urology, Fuzong Clinical Medical College, Fujian Medical University, Fuzhou, China.
Department of Nephrology, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, China.
RNA Biol. 2025 Dec;22(1):1-12. doi: 10.1080/15476286.2024.2448387. Epub 2025 Jan 5.
Mutations in coding sequence and abnormal PKD1 expression levels contribute to the development of autosomal-dominant polycystic kidney disease, the most common genetic disorder. Regulation of PKD1 expression by factors located in the promoter and 3´ UTR have been extensively studied. Less is known about its regulation by 5´ UTR elements. In this study, we investigated the effects of uORFs and uORF-affecting variants by combining bioinformatic analyses, luciferase reporter assays, RT-qPCR and immunoblotting experiments. Our analyses demonstrate that mRNA contains two evolutionarily conserved translation-inhibitory uORFs. uORF1 is translatable, and uORF2 is likely not translatable. The 5´ UTR and uORFs do not modulate downstream protein output under endoplasmic reticulum stress and oxidative stress conditions. Some of uORF-perturbing variants in the SNP database are predicted to affect gene translation. Luciferase reporter assays and RT-qPCR results reveal that rs2092942382 and rs1596636969 increase, while rs2092942900 decreases main gene translation without affecting transcription. Antisense oligos targeting the uORFs reduce luciferase protein levels without altering luciferase mRNA levels. Our results establish as a novel target of uORF-mediated translational regulation and mutations that perturb uORFs may dysregulate PKD1 protein level.
编码序列中的突变和异常的PKD1表达水平促成了常染色体显性多囊肾病的发展，这是最常见的遗传性疾病。位于启动子和3´UTR的因子对PKD1表达的调控已得到广泛研究。而关于其受5´UTR元件调控的了解较少。在本研究中，我们通过结合生物信息学分析、荧光素酶报告基因检测、RT-qPCR和免疫印迹实验，研究了上游开放阅读框（uORF）和影响uORF的变异体的作用。我们的分析表明，mRNA包含两个进化上保守的翻译抑制性uORF。uORF1是可翻译的，而uORF2可能不可翻译。在 内质网应激和氧化应激条件下，5´UTR和uORF不会调节下游蛋白质的产出。SNP数据库中一些干扰uORF的变异体预计会影响基因翻译。荧光素酶报告基因检测和RT-qPCR结果显示，rs2092942382和rs1596636969会增加，而rs2092942900会降低主要基因的翻译，且不影响转录。靶向uORF的反义寡核苷酸可降低荧光素酶蛋白水平，而不改变荧光素酶mRNA水平。我们的结果确定了PKD1是uORF介导的翻译调控的新靶点，干扰uORF的突变可能会使PKD1蛋白水平失调。
