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SUPREM:一种工程化非站点特定的m6ARNA甲基转移酶,具有高度提高的效率

Yoshiki Ochiai¹, Ben E Clifton¹, Madeleine Le Coz²

¹Protein Engineering and Evolution Unit, Okinawa Institute of Science and Technology Graduate University (OIST), 1919-1 Tancha, Onna, Kunigami District, Okinawa 904-0495, Japan.

Nucleic acids research

|October 17, 2024

概括

研究人员设计了一种新型酶,SUPER RNA EcoGII 甲基转移酶 (SUPREM),用于增强N 6-甲基亚丁 (m6A) RNA甲基化. 这种工具显示出更大的活性和稳定性,推动了RNA修饰研究.

科学领域:

生物化学生物化学
分子生物学分子生物学
合成生物学合成生物学

背景情况:

N6-甲基氨酸 (m6A) RNA甲基化对于细胞过程至关重要,但很难在个别修饰部位进行研究.
目前的RNA甲基化研究在分离特定的m6A修饰方面面临着挑战.
工程RNA甲基转移酶 (RNA MTases) 对于开发精确的合成生物学工具来进行RNA操纵至关重要.

研究的目的:

设计一种更有效,更稳定的RNA甲基转移酶,用于研究N6-甲基亚丁 (m6A) RNA修饰.
探索细菌DNA甲基转移酶EcoGII (M.EcoGII) 的潜力,用于针对RNA甲基化的蛋白质工程.
开发改进的合成生物学工具,用于体内RNA甲基化和标记.

主要方法:

祖先序列重建以探索M.EcoGII.的序列空间.
蛋白质工程创造了SUPER RNA EcoGII 甲基转移酶 (SUPREM).
在哺乳动物细胞中使用免疫光,LC-MS/MS和纳米孔直接RNA测序对SUPREM和M.EcoGII进行比较分析.

主要成果:

与M.EcoGII相比,SUPREM的表达率提高了8倍,温度稳定性提高了7°C,m6ARNA甲基化活性增加了12倍.
在哺乳动物细胞中,SUPREM显示出显著更高的RNA甲基化活性,并针对更广泛的RNA甲基化位点.
遗传学和突变分析确定了一个关键的残留物,负责SUPREM增强的RNA甲基化活性.

结论:

SUPREM是一种高效,非特异性RNA甲基转移酶,其性能比M.EcoGII.II更好.
工程 SUPREM 酶为先进的 in vivo RNA 甲基化和标签应用提供了一个有前途的工具.
这项研究强调了通过祖先序列重建来开发新型合成生物学工具的蛋白质工程的潜力.

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