Enzymatic or In Vivo Installation of Propargyl Groups in Combination with Click Chemistry for the Enrichment and Detection of Methyltransferase Target Sites in RNA,Angewandte Chemie International Edition

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Enzymatic or In Vivo Installation of Propargyl Groups in Combination with Click Chemistry for the Enrichment and Detection of Methyltransferase Target Sites in RNA
Angewandte Chemie International Edition ( IF 16.1 ) Pub Date : 2018-03-22 , DOI: 10.1002/anie.201800188
Katja Hartstock ₁ , Benedikt S. Nilges ₂ , Anna Ovcharenko ₁ , Nicolas V. Cornelissen ₁ , Nikolai Püllen ₁ , Ann-Marie Lawrence-Dörner ₁ , Sebastian A. Leidel ₂ , Andrea Rentmeister ₁

Affiliation

m⁶A is the most abundant internal modification in eukaryotic mRNA. It is introduced by METTL3‐METTL14 and tunes mRNA metabolism, impacting cell differentiation and development. Precise transcriptome‐wide assignment of m⁶A sites is of utmost importance. However, m⁶A does not interfere with Watson–Crick base pairing, making polymerase‐based detection challenging. We developed a chemical biology approach for the precise mapping of methyltransferase (MTase) target sites based on the introduction of a bioorthogonal propargyl group in vitro and in cells. We show that propargyl groups can be introduced enzymatically by wild‐type METTL3‐METTL14. Reverse transcription terminated up to 65 % at m⁶A sites after bioconjugation and purification, hence enabling detection of METTL3‐METTL14 target sites by next generation sequencing. Importantly, we implemented metabolic propargyl labeling of RNA MTase target sites in vivo based on propargyl‐l‐selenohomocysteine and validated different types of known rRNA methylation sites.

中文翻译：

酶促或体内制备炔丙基与单击化学的结合，用于富集和检测RNA中的甲基转移酶靶位

m ⁶ A是真核mRNA中最丰富的内部修饰。它是由METTL3-METTL14引入的，可调节mRNA的新陈代谢，影响细胞的分化和发育。在整个转录组中精确分配m ⁶ A位点至关重要。但是，m ⁶ A不会干扰Watson-Crick碱基配对，这使基于聚合酶的检测具有挑战性。我们在体外和细胞中引入了生物正交炔丙基的基础上，开发了一种化学生物学方法，用于精确定位甲基转移酶（MTase）目标位点。我们显示，野生型METTL3-METTL14可以通过酶法引入炔丙基。在m ⁶处反转录终止率高达65％生物缀合和纯化后的一个位点，因此可以通过下一代测序检测METTL3-METTL14目标位点。重要的是，我们基于炔丙基-1-硒代同型半胱氨酸在体内对RNA MTase目标位点进行了代谢炔丙基标记，并验证了不同类型的已知rRNA甲基化位点。

更新日期：2018-03-22

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