金属离子结合和谷氨酰胺供应影响tRNAGln的解码效率

doi:10.5246/jcps.2025.01.003

中国药学(英文版) ›› 2025, Vol. 34 ›› Issue (1): 28-40.DOI: 10.5246/jcps.2025.01.003

金属离子结合和谷氨酰胺供应影响tRNAGln的解码效率

沈雨轩¹^,^#, 王天畅²^,^#, 乔桦², 梁庆², 吕靖茹², 夏青¹^,²^,^*()

^1. 北京大学医学部医学技术研究院, 北京 100191
^2. 北京大学药学院分子与细胞药理学系, 北京 100191

收稿日期:2024-09-21 修回日期:2024-10-11 接受日期:2024-11-23 出版日期:2025-02-20 发布日期:2025-02-20
通讯作者: 夏青

Modulation of tRNAGln decoding efficacy by metal ion binding and glutamine supply

Yuxuan Shen¹^,^#, Tianchang Wang²^,^#, Hua Qiao², Qing Liang², Jingru Lv², Qing Xia¹^,²^,^*()

¹ Institute of Medical Technology, Peking University Health Science Center, Beijing 100091, China
² Department of Molecular and Cellular Pharmacology, School of Pharmaceutical Sciences, Peking University, Beijing 100091, China

Received:2024-09-21 Revised:2024-10-11 Accepted:2024-11-23 Online:2025-02-20 Published:2025-02-20
Contact: Qing Xia
About author:
^# Yuxuan Shen and Tianchang Wang contributed equally to this work.
Supported by:
National Natural Science Foundation of China (Grant No. U23A20106), National Key Research and Development Program of China (Grant No. 91510100MA6CG8UJ4K).

摘要/Abstract

摘要：

tRNAs需要形成稳定的L-型三级结构以参与到蛋白质的翻译过程中。二价金属离子, 尤其是镁离子, 对维持tRNA三级结构的稳定性十分重要。但对于人tRNA中Mg²⁺的结合位点至今仍不清楚。在本次研究中, 为确定Mg²⁺在人tRNA^Gln中的结合位点, 将能够识别提前终止密码子UAG, 促进全长蛋白表达的抑制性tRNA作为研究工作。我们突变了抑制性tRNA^Gln的U8和C72位点, 并检测突变后的抑制性tRNA通读效率, 以通读效率表示tRNA的解码能力。通过分别检测U8C和C72U抑制性tRNA突变体中Mg²⁺的含量, 发现U8C突变体中的Mg²⁺含量显著低于C72U突变体。进一步, 通过GFP荧光和Western Blot实验, 证实了与野生型抑制性tRNA相比, U8位突变体对无义突变位点的通读能力显著降低。然而, 抑制性tRNA C72位的突变对通读效率的影响明显低于U8位。这些结果证实, Mg²⁺与人tRNA^GlnU8位点紧密结合, 维持tRNA三维结构的稳定性以及其蛋白翻译能力。此外, 我们研究了谷氨酰胺供应量对细胞水平 tRNA 解码效率的影响, 结果表明, 氨基酸浓度和TAG密码子上下文都会影响 RNA 的解码效率。本研究为进一步探究tRNA的结构和功能提供了理论基础。

关键词: 金属离子, tRNA三级结构, 谷氨酰胺供应, 解码效率

Abstract:

Transfer RNAs (tRNAs) adopt a stable L-shaped tertiary structure crucial for their involvement in protein translation. Among various divalent metal ions, magnesium ions play a pivotal role in preserving the tertiary structure of tRNA. However, the precise location of the Mg²⁺ binding pocket in human tRNA remains elusive. In this investigation, we identified the Mg²⁺ binding site within human tRNA^Gln using suppressor tRNA^Gln. This variant of tRNA recognizes premature stop codons (specifically UAG) and facilitates the expression of full-length proteins. By mutating sites U8 and C72 in suppressor tRNA^Gln, we assessed the decoding efficiency of the resulting mutant suppressor tRNAs, which serves as a measure of tRNA’s ability to decode genetic information. Our analysis revealed that the U8C mutant suppressor tRNA exhibited a significantly lower Mg²⁺ content compared to the C72U mutant. Furthermore, we observed a notable reduction in decoding efficiency in the U8-mutated suppressor tRNA, as evidenced by GFP fluorescence and Western blotting analysis. Conversely, mutations at the C72 site had a comparatively minor impact on decoding efficiency. These findings underscored the tight binding of Mg²⁺ to the U8 site of human tRNA^Gln, crucial for maintaining the stability of tRNA tertiary structure and translation efficacy. Additionally, our investigation delved into the influence of glutamine availability on tRNA decoding efficiency at the cellular level. The results indicated that both the concentration of amino acids and the codon context of TAG could modulate tRNA decoding efficiency. This study provided valuable insights into the structure and function of tRNA, laying the groundwork for further exploration in this field.

Key words: Metal ions, tRNA tertiary structure, Glutamine supply, Decoding efficacy

Supporting:

沈雨轩, 王天畅, 乔桦, 梁庆, 吕靖茹, 夏青. 金属离子结合和谷氨酰胺供应影响tRNAGln的解码效率[J]. 中国药学(英文版), 2025, 34(1): 28-40.

Yuxuan Shen, Tianchang Wang, Hua Qiao, Qing Liang, Jingru Lv, Qing Xia. Modulation of tRNAGln decoding efficacy by metal ion binding and glutamine supply[J]. Journal of Chinese Pharmaceutical Sciences, 2025, 34(1): 28-40.

图/表 6

Figure 1. stRNA construction. (A) Single-base substitution of tRNAGln anticodon to obtain stRNAGln. (B) Cloning three types of Pol III promoters, 7SK, hU6, and H1, upstream of stRNAGln.

Figure 2. stRNA promoter comparison. (A) Dual luciferase TAG report system construction. (B) GFP-TAG report system construction. (C) Decoding efficiency of stRNAGln promoted by 7SK, hU6, and H1 promoters, measured by detecting the ratio of firefly and renilla chemiluminescence via dual-luciferase assay (n = 3). Error bars denote SD. *P < 0.05; **P < 0.01 (one-way analysis of variance (ANOVA) with Newman-Keuls multiple comparisons test). (D) GFPTAG fluorescence intensity detected after transfecting stRNAs with various promoters.

Figure 3. Content of Mg2+ in mutant stRNA. (A) Mutation of stRNA at Mg2+ binding sites in yeast tRNAPhe. (B) Total tRNA separated from total RNA extracted from HEK293T cells transfected with U8 or C72 mutated stRNAs and nonsense mutation GFP by 8% urea-PAGE. (C) Content of Mg2+ in 1 µg tRNA of U8C and C72U mutant stRNA groups (n = 3). ***P < 0.001 (ANOVA with Newman-Keuls multiple comparisons test).

Figure 4. Decoding efficiency of U8 mutant stRNAGln. (A) GFP fluorescence intensity detected after transfecting U8C, U8G and U8A mutant stRNAGln. (B) Decoding efficiency of mutant stRNAGln measured by detecting the ratio of firefly and renilla chemiluminescence via dual-luciferase assay (n = 3). Error bars denote SD. ***P < 0.001 (ANOVA with Newman-Keuls multiple comparisons test). (C) Effect of U8C, U8G and U8A mutation in stRNAGln on GFPTAG expression detected by Western blotting assay. (D) The binding of Mg2+ with U8 site in tertiary structure of human tRNAGln.

Figure 5. Decoding efficiency of C72 mutant stRNAGln. (A) GFP fluorescence intensity detected after transfecting C72A, C72U, and C72G mutant stRNAGln. (B) Decoding efficiency of mutant stRNAGln measured by detecting the ratio of firefly and renilla chemiluminescence via dual-luciferase assay (n = 3). Error bars denote SD. ***P < 0.001 (ANOVA with Newman-Keuls multiple comparisons test); n.s., not significant. (C) Effect of C72A, C72U and C72G mutation in stRNAGln on GFPTAG expression detected by Western blotting assay.

Figure 6. Varying concentrations of amino acids influence the decoding efficiency of stRNAGln. (A) Plasmid construction of optimized and DMD patient-derived codon context of TAG in the dual-luciferase system. (B) Decoding efficiency of stRNAGln treated with varying concentrations of glutamine, measured by detecting the ratio of firefly and renilla chemiluminescence via dual-luciferase assay (n = 3). Error bars denote SD. *P < 0.05; **P < 0.01 (ANOVA with Newman-Keuls multiple comparisons test). (C) The efficiency of stRNAGln decoding TAG from two DMD patients treated with varying concentrations of glutamine, measured by detecting the ratio of firefly and renilla chemiluminescence via dual-luciferase assay (n = 3). Error bars denote SD. *P < 0.05; **P < 0.01 (ANOVA with Newman-Keuls multiple comparisons test).

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金属离子结合和谷氨酰胺供应影响tRNAGln的解码效率

Modulation of tRNAGln decoding efficacy by metal ion binding and glutamine supply

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