Mapping N6 -Methyladenosine (m6 A) in RNA: Established Methods, Remaining Challenges, and Emerging Approaches.

N6 -Methyladenosine (m6 A) is the most abundant internal modification in eukaryotic mRNA. Specific m6 A 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 m6 A and mechanisms governing its regulation. In this Minireview, we summarize established and emerging concepts for m6 A mapping. Starting with the seminal m6 A-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 m6 A-sensitive enzymes and chemical biology approaches combining substrate analogues, chemical derivatization, and enzymatic steps to trace m6 A. Finally, single-molecule sequencing as a new avenue for direct detection of mRNA modifications will be discussed.

Enzymatic or In Vivo Installation of Propargyl Groups in Combination with Click Chemistry for the Enrichment and Detection of Methyltransferase Target Sites in RNA.

m6 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 m6 A sites is of utmost importance. However, m6 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 m6 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.

MePMe-seq: antibody-free simultaneous m6A and m5C mapping in mRNA by metabolic propargyl labeling and sequencing.

Internal modifications of mRNA have emerged as widespread and versatile regulatory mechanism to control gene expression at the post-transcriptional level. Most of these modifications are methyl groups, making S-adenosyl-L-methionine (SAM) a central metabolic hub. Here we show that metabolic labeling with a clickable metabolic precursor of SAM, propargyl-selenohomocysteine (PSH), enables detection and identification of various methylation sites. Propargylated A, C, and G nucleosides form at detectable amounts via intracellular generation of the corresponding SAM analogue. Integration into next generation sequencing enables mapping of N6-methyladenosine (m6A) and 5-methylcytidine (m5C) sites in mRNA with single nucleotide precision (MePMe-seq). Analysis of the termination profiles can be used to distinguish m6A from 2'-O-methyladenosine (Am) and N1-methyladenosine (m1A) sites. MePMe-seq overcomes the problems of antibodies for enrichment and sequence-motifs for evaluation, which was limiting previous methodologies. Metabolic labeling via clickable SAM facilitates the joint evaluation of methylation sites in RNA and potentially DNA and proteins.

Chemoenzymatic labeling of RNA to enrich, detect and identify methyltransferase-target sites.

The RNA methyltransferase (MTase) complex METTL3-METTL14 transfers methyl groups from S-adenosyl-l-methionine (AdoMet) to the N6-position of adenosines within its consensus sequence, the DRACH motif (D=A, G, U; R=A, G; H=A, C, U). Interestingly, this MTase complex shows remarkable promiscuity regarding the cosubstrate. This can be exploited to install nonnatural modifications, like clickable or photocaging groups. Clickable groups are widely used for subsequent functionalization and open a broad range of possibilities for downstream applications. Here, we elaborate on click chemistry for coupling of RNA to biotin to enrich MTase targets via streptavidin-coated magnetic beads. Importantly, after clicking and coupling to beads the modification becomes sterically demanding and stalls reverse transcriptases, leading to termination adjacent to the MTase target site. Using radioactively labeled primers in the reverse transcription, the modified position can be precisely identified on a sequencing gel via phosphor imaging.

Chemo-enzymatic treatment of RNA to facilitate analyses.

Labeling RNA is a recurring problem to make RNA compatible with state-of-the-art 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 RNA-modifying 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 chemo-enzymatic 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. This article is categorized under: RNA Processing < RNA Editing and Modification RNA Methods < RNA Analyses in vitro and In Silico RNA Methods < RNA Analyses in Cells.