Kim Yun Ji, Hong So-Hee
Department of Microbiology, Ewha Womans University College of Medicine, Seoul, Korea.
Ewha Med J. 2025 Jul;48(3):e42. doi: 10.12771/emj.2025.00423. Epub 2025 May 27.
Internal ribosome entry site (IRES) elements, present in both viral and cellular messenger RNAs (mRNAs), facilitate cap-independent translation by recruiting ribosomes to internal regions of mRNA. This study aimed to investigate the impact of inserting G-quadruplex and hairpin structures into the 5' untranslated region (UTR) and poly(A) sequences on the translation efficiency of the encephalomyocarditis virus (EMCV) IRES, using an IRES-based RNA platform encoding OX40L, 4-1BBL, and GFP.
G-quadruplex and hairpin structures, derived from HIV-1 (human immunodeficiency virus type 1) or custom-designed, were synthesized and inserted into the 5' UTR and poly(A) tail regions of EMCV IRES vectors. These constructs were amplified by polymerase chain reaction, ligated into plasmids, and transcribed in vitro. B16 melanoma, TC-1 tumor, and HEK293 cells were transfected with these RNA constructs. Protein expression levels were assessed at 6, 12, and 24 hours post-transfection by flow cytometry and fluorescence microscopy. Statistical analyses employed one-way analysis of variance with the Dunnett test.
The insertion of G-quadruplex and hairpin structures altered RNA secondary structure, significantly reducing protein expression. In the 5' UTR, the G-quadruplex nearly abolished OX40L expression (1.18%±0.41% at 6 hours vs. 18.23%±0.16% for control), while the hairpin structure reduced it (16.29%±1.46% vs. 22.84%±1.17%). In the poly(A) tail region, both structures decreased GFP expression across all cell lines (4.86%±1.35% to 7.27%±0.32% vs. 39.56%±2.07% in B16 cells).
Inserting G-quadruplex and hairpin structures into EMCV IRES UTRs inhibits translation efficiency, suggesting the need for precise RNA structure modeling to enhance IRES-mediated translation.
内部核糖体进入位点（IRES）元件存在于病毒和细胞信使核糖核酸（mRNA）中，通过将核糖体招募到mRNA的内部区域来促进不依赖帽结构的翻译。本研究旨在利用编码OX40L、4-1BBL和绿色荧光蛋白（GFP）的基于IRES的RNA平台，研究在5'非翻译区（UTR）和多聚腺苷酸（poly(A)）序列中插入G-四链体和发夹结构对脑心肌炎病毒（EMCV）IRES翻译效率的影响。
合成源自1型人类免疫缺陷病毒（HIV-1）或定制设计的G-四链体和发夹结构，并将其插入EMCV IRES载体的5'UTR和poly(A)尾区域。这些构建体通过聚合酶链反应进行扩增，连接到质粒中，并进行体外转录。用这些RNA构建体转染B16黑色素瘤细胞、TC-1肿瘤细胞和人胚肾293（HEK293）细胞。在转染后6小时、12小时和24小时，通过流式细胞术和荧光显微镜评估蛋白质表达水平。统计分析采用单因素方差分析和Dunnett检验。
G-四链体和发夹结构的插入改变了RNA二级结构，显著降低了蛋白质表达。在5'UTR中，G-四链体几乎消除了OX40L的表达（6小时时为1.18%±0.41%，而对照为18.23%±0.16%），而发夹结构降低了其表达（16.29%±1.46%对22.84%±1.17%）。在poly(A)尾区域，两种结构均降低了所有细胞系中的GFP表达（B16细胞中为4.86%±1.35%至7.27%±0.32%，而对照为39.56%±2.07%）。
在EMCV IRES的UTR中插入G-四链体和发夹结构会抑制翻译效率，这表明需要进行精确的RNA结构建模以增强IRES介导的翻译。
