Functional studies of the RNA N6-methyladenosine (m6A) modification have been limited by an inability to map individual m6A-modified sites in whole transcriptomes. To enable such studies, University of Chicago researchers have developed m6A-selective allyl chemical labeling and sequencing (m6A-SAC-seq), a method for quantitative, whole-transcriptome mapping of m6A at single-nucleotide resolution. The method requires only ~30 ng of poly(A) or rRNA-depleted RNA. The researchers mapped m6A modification stoichiometries in RNA from cell lines and during in vitro monocytopoiesis from human hematopoietic stem and progenitor cells (HSPCs). They identified numerous cell-state-specific m6A sites whose methylation status was highly dynamic during cell differentiation. They observed changes of m6A stoichiometry as well as expression levels of transcripts encoding or regulated by key transcriptional factors (TFs) critical for HSPC differentiation. m6A-SAC-seq is a quantitative method to dissect the dynamics and functional roles of m6A sites in diverse biological processes using limited input RNA.
m6A-SAC-seq strategy and development
MjDim1 uses allylic-SAM as a cofactor to label m6A to a6m6A, which undergoes cyclization following I2 treatment. b, An m6A-modified 12-mer RNA probe was treated with MjDim1 and allylic-SAM, followed by matrix-assisted laser desorption ionization (MALDI) characterization. The added molecular weight is that of the allyl group. c, An m6A-free 12-mer RNA probe was treated with MjDim1 and allylic-SAM, followed by MALDI characterization. No detectable new product appeared. d, Michaelis–Menten steady-state kinetics of the MjDim1-catalyzed allyl transfer to an m6A-containing probe (MALDI_Probe_m6A in Supplementary Table 1). Data are represented as mean ± s.e.m. for two biological replicates × two technical replicates. e, Michaelis–Menten steady-state kinetics of the MjDim1-catalyzed allyl transfer to an unmodified control probe (MALDI_Probe_A in Supplementary Table 1). Data are represented as mean ± s.e.m. for two biological replicates × two technical replicates. f, Cyclized a6m6A induces higher mutation rates than cyclized a6A in various RNA sequence contexts when using HIV RT. RNA oligonucleotides containing a6A or a6m6A were synthesized by incorporating O6-phenyl-adenosine phosphoramidite into the designed sequence containing an NNXNN motif (X = a6A or a6m6A).