Hartstock Katja, Nilges Benedikt S, Ovcharenko Anna, Cornelissen Nicolas V, Püllen Nikolai, Lawrence-Dörner Ann-Marie, Leidel Sebastian A, Rentmeister Andrea
Institute of Biochemistry, Department of Chemistry, University of Münster, Wilhelm-Klemm-Straße 2, 48149, Münster, Germany.
Max Planck Research Group for RNA Biology, Max Planck Institute for Molecular Biomedicine, Röntgenstraße 20, 48149, Münster, Germany.
Angew Chem Int Ed Engl. 2018 May 22;57(21):6342-6346. doi: 10.1002/anie.201800188. Epub 2018 Mar 22.
m A 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 m A sites is of utmost importance. However, m A 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 m A 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.
N⁶-甲基腺嘌呤（m⁶A）是真核生物信使核糖核酸（mRNA）中最丰富的内部修饰。它由甲基转移酶样3（METTL3）-甲基转移酶样14（METTL14）引入，并调节mRNA代谢，影响细胞分化和发育。精确确定全转录组范围的m⁶A位点至关重要。然而，m⁶A并不干扰沃森-克里克碱基配对，这使得基于聚合酶的检测具有挑战性。我们开发了一种化学生物学方法，用于基于在体外和细胞中引入生物正交炔丙基来精确绘制甲基转移酶（MTase）的靶位点。我们表明，野生型METTL3-METTL14可以通过酶促反应引入炔丙基。在生物共轭和纯化后，逆转录在m⁶A位点处终止的比例高达65%，从而能够通过下一代测序检测METTL3-METTL14的靶位点。重要的是，我们基于炔丙基-L-硒代高半胱氨酸在体内实现了RNA甲基转移酶靶位点的代谢炔丙基标记，并验证了不同类型的已知核糖体RNA（rRNA）甲基化位点。
