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

Abstract

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.

Mapping N6‐Methyladenosine (m6A) in RNA: Established Methods, Remaining Challenges, and Emerging Approaches

“…The RNA is isolated from cells and fragmented, then the propargyl groups are reacted by copper‐catalyzed azide–alkyne cycloaddition (CuAAC) with biotin azide, followed by enrichment with streptavidin‐coated magnetic beads (SA). Reverse transcription (RT) and subsequent next‐generation sequencing (NGS) allowed identification of MTase target sites …”

“…Propargyl‐ l ‐selenohomo‐cysteine ( 5 ) is taken up by cells and converted to SeAdoYn ( 6 ), which is then used by MTases as cosubstrate to install a propargyl group at their RNA target sites . The propargyl modification of adenosine in RNA could be confirmed by LC‐MS . To date, the approach was used to identify known MTase target sites, specifically 2′‐ O ‐methylations, in 18S rRNA based on enrichment, and future work will reveal whether m 6 A sites can be detected and if the strategy can be applied to mRNA .…”

“…The propargyl modification of adenosine in RNA could be confirmed by LC‐MS . To date, the approach was used to identify known MTase target sites, specifically 2′‐ O ‐methylations, in 18S rRNA based on enrichment, and future work will reveal whether m 6 A sites can be detected and if the strategy can be applied to mRNA . Since AdoMet serves as cosubstrate for many MTases, this approach bears potential to detect various MTase target sites at once, although this is likely to complicate the RT signatures.…”

“…Allyl groups were transferred to the N 6 ‐position of unmethylated adenosines in RNA isolated from HeLa cells . The conversion was significantly lower than propargyl transfer from the respective cosubstrate SeAdoYn . The N 6 ‐allyladenosine was used for subsequent iodination under mild buffer conditions, followed by reaction between the iodinated alkyl chain with the N 1‐position of adenosine leading to the formation of N 1‐ N 6 ‐cyclized adenosine.…”

“…[61] Recently,w es howed that recombinant METTL3-METTL14 also efficiently transfers ap ropargyl group to the N 6 -position of adenosine within the consensus motif of at arget RNA, if the respective selenium-based cosubstrate analogue (SeAdoYn, 6) is used. [59] The N 6 -propargyl group itself only slightly increased termination during RT,b ut allowed for bioconjugation with biotin-azide by ac opper-catalyzed alkyne-azide cycloaddition (CuAAC). The click product didn ot only increase the steric demand of the modification but also allowed immobilization on streptavidin-coated magnetic beads (Figure 4).…”