Cornelissen, N. V. | bioRxiv Science

bioRxiv2022

Biomimetic S-adenosylmethionine regeneration for nucleophilic and radical alkylation reactions and aminopropyltransfer

S-Adenosylmethionine (SAM) is an enzyme cofactor involved in methylation, aminopropyl transfer, and radical reactions. This versatility renders SAM-dependent enzymes of great interest in biocatalysis. The usage of SAM analogues adds to this diversity. However, high cost and instability of the cofactor impedes the investigation and usage of these enzymes. While SAM regeneration protocols from the methyltransferase (MT) byproduct S-adenosylhomocysteine are available, aminopropyl transferases and radical SAM enzymes are not covered. Here, we report an efficient one-pot system to supply or regenerate SAM and SAM analogues for all three enzyme classes. The systems flexibility is showcased by the transfer of an ethyl group with a cobalamin-dependent radical SAM MT using S-adenosylethionine as a cofactor. This shows the potential of SAM (analogue) supply and regeneration for the application of diverse chemistry, as well as for mechanistic studies using cofactor analogues.

biochemistry↗

bioRxiv2022

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. 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.

biochemistry↗