m⁶A-SAC-seq: m⁶A RNA modifications measured at single-base resolution across the mammalian transcriptome

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).