Institute of Biochemistry, University of Münster, Wilhelm-Klemm-Straße 2, 48149 Münster, Germany.
Cells-in-Motion Cluster of Excellence (EXC1003-CiM), University of Münster, Germany.
Nucleic Acids Res. 2019 Apr 23;47(7):e42. doi: 10.1093/nar/gkz084.
Post-transcriptional regulation of gene expression occurs by multiple mechanisms, including subcellular localization of mRNA and alteration of the poly(A) tail length. These mechanisms play crucial roles in the dynamics of cell polarization and embryonic development. Furthermore, mRNAs are emerging therapeutics and chemical alterations to increase their translational efficiency are highly sought after. We show that yeast poly(A) polymerase can be used to install multiple azido-modified adenosine nucleotides to luciferase and eGFP-mRNAs. These mRNAs can be efficiently reacted in a bioorthogonal click reaction with fluorescent reporters without degradation and without sequence alterations in their coding or untranslated regions. Importantly, the modifications in the poly(A) tail impact positively on the translational efficiency of reporter-mRNAs in vitro and in cells. Therefore, covalent fluorescent labeling at the poly(A) tail presents a new way to increase the amount of reporter protein from exogenous mRNA and to label genetically unaltered and translationally active mRNAs.
基因表达的转录后调控是通过多种机制实现的，包括 mRNA 的亚细胞定位和聚（A）尾长度的改变。这些机制在细胞极化和胚胎发育的动力学中起着至关重要的作用。此外，mRNA 正在成为治疗方法，并且人们强烈追求通过化学修饰来提高其翻译效率。我们表明，酵母多聚（A）聚合酶可用于向荧光素酶和 eGFP-mRNA 中安装多个叠氮修饰的腺苷核苷酸。这些 mRNA 可以在生物正交点击反应中与荧光报告物有效地反应，而不会降解，并且其编码或非翻译区没有序列改变。重要的是，聚（A）尾的修饰对体外和细胞内报告基因-mRNA 的翻译效率有积极的影响。因此，在聚（A）尾上进行共价荧光标记为从外源 mRNA 中增加报告蛋白的量并标记遗传上未改变且翻译活性的 mRNA 提供了一种新方法。
