Multiple covalent fluorescence labeling of eukaryotic mRNA at the poly(A) tail enhances translation and can be performed in living cells
Multiple covalent fluorescence labeling of eukaryotic mRNA at the poly(A) tail enhances translation and can be performed in living cells
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DOI：
10.1093/nar/gkz084
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发表时间：
2019-04-23
影响因子：
14.9
通讯作者：
Rentmeister, Andrea
中科院分区：
文献类型：
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作者：
Anhaeuser, Lea;Huewel, Sabine;Rentmeister, Andrea
基因表达的转录后调节通过多种机制发生，包括mRNA的亚细胞定位和poly（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是新兴的治疗剂，并且高度寻求增加其翻译效率的化学改变。我们表明，酵母聚（A）聚合酶可用于安装多个叠氮基修饰的腺苷核苷酸荧光素酶和eGFP-mRNA。这些mRNA可以在生物正交点击反应中与荧光报告分子有效地反应，而不会降解，并且在其编码区或非翻译区中没有序列改变。重要的是，poly（A）尾中的修饰对体外和细胞中转录因子-mRNA的翻译效率产生积极影响。因此，在poly（A）尾处的共价荧光标记提供了一种新的方式来增加来自外源mRNA的报告蛋白的量，并标记遗传上未改变的和具有抑制活性的mRNA。
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.