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.

Labeling RNA is a recurring problem to make RNA compatible with state-of-theart methodology and comes in many flavors. Considering only cellular applications, the spectrum still ranges from site-specific labeling of individual transcripts, for example, for live-cell imaging of mRNA trafficking, to metabolic labeling in combination with next generation sequencing to capture dynamic aspects of RNA metabolism on a transcriptome-wide scale. Combining the specificity of RNAmodifying enzymes with non-natural substrates has emerged as a valuable strategy to modify RNA site-or sequence-specifically with functional groups suitable for subsequent bioorthogonal reactions and thus label RNA with reporter moieties such as affinity or fluorescent tags. In this review article, we will cover chemoenzymatic approaches (a) for in vitro labeling of RNA for application in cells, (b) for treatment of total RNA, and (c) for metabolic labeling of RNA.

N6‐Methyladenosine (m6A) is the most abundant internal modification in eukaryotic mRNA. Specific m6A reader and eraser proteins link this modification to many aspects of mRNA metabolism and regulate its levels in a dynamic way. Precise localization and quantification in varying biological samples is, therefore, relevant to understand the functional role of m6A and mechanisms governing its regulation. In this Minireview, we summarize established and emerging concepts for m6A mapping. Starting with the seminal m6A‐sequencing techniques based on immunoprecipitation, we will highlight technical improvements by photo‐cross‐linking and remaining challenges. As an alternative, antibody‐free approaches will be presented. These include wild‐type or engineered m6A‐sensitive enzymes and chemical biology approaches combining substrate analogues, chemical derivatization, and enzymatic steps to trace m6A. Finally, single‐molecule sequencing as a new avenue for direct detection of mRNA modifications will be discussed.

Internal modifications of mRNA have emerged as widespread and versatile regulatory mechanism to control gene expression at the post-transcriptional level. Current insights rely on the ability to make a modified nucleoside amenable to sequencing. Most of the modifications are methylations involving the co-factor S-adenosyl-L-methionine (SAM), however, simultaneous detection of different methylation sites in the same sample has remained elusive. We present metabolic labeling with propargyl-selenohomocysteine (PSH) in combination with click chemistry to detect N6-methyladenosine (m6A) and 5-methylcytidine (m5C) sites in mRNA with single nucleotide precision in the same sequencing run (MePMe-seq). Intracellular formation of the corresponding SAM analogue leads to detectable levels of N6-propargyl-A (prop6A) and 5-propargyl-C (prop5C). MePMe-seq overcomes the problems of antibodies for enrichment and sequence-motifs for evaluation, limiting previous methodologies. The joint evaluation of m6A and m5C sites opens the door to study their interconnectivity and improve our understanding of mechanisms and functions of the RNA methylome.

Die präzise,transkriptomweite Kartierung von m 6 A-Positionen, einer der häufigsten internen Modifikationen in eukaryotischer mRNA, ist von großem Interesse.E ine Polymerase-basierte Detektion von m 6 Ai st schwierig, da die Modifikation keinen Einfluss auf die Watson-Crick-Basenpaarung hat. Ein chemisch-biologischer Ansatz wurde entwickelt, der die präzise Kartierung von Zielsequenzen von Methyltransferasen (MTase) durch Einbau einer bioorthogonalen Propargylgruppe in vitro und in Zellen ermçglicht. Die Propargylgruppe wird hierbei enzymatisch von Wildtyp-METTL3-METTL14 angebracht. Nach Biokonjugation und Reinigung brichtd ie reverse Transkription an den m 6 A-Positionen in 65 %der Fälle ab.Dies ermçglicht die Detektion der METTL3-METTL14-Zielsequenzen durchS equenzierungsmethoden der nächsten Generation. Der metabolische Einbau von Propargylgruppen in MTase-Zielsequenzen gelang auch in vivo mithilfe von Propargyl-l-selenohomocystein;s o konnten bekannte rRNA-Methylierungsstellen validiert werden.

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Ein präziser Kartierungsansatz zur Detektion von Zielstrukturen von RNA‐Methyltransferase wird von A. Rentmeister et al. in ihrer Zuschrift auf S. 6451 ff. beschrieben. Eine bioorthogonale Propargylgruppe wird enzymatisch auf RNA übertragen, und die Methyltransferase‐Zielstrukturen können im Anschluss mittels Biokonjugation, Anreicherung sowie Sequenzierungsmethoden der nächsten Generation detektiert werden. Das Bild zeigt die Kartierung eines RNA‐Waldes durch Einführung einer Propargylgruppe zur Unterscheidung zwischen modifizierten und nichtmodifizierten Adenosinen.

A precise mapping approach to detect RNA methyltransferase target sites is described by A. Rentmeister et al. in their Communication on page 6342 ff. A bioorthogonal propargyl group is enzymatically transferred to RNA, and the methyltransferase target sites can then be detected through bioconjugation, enrichment, and next‐generation sequencing. The picture shows the mapping of an RNA forest through the introduction of a propargyl group to distinguish between modified and non‐modified adenosines.