Current progress in strategies to profile transcriptomic m⁶A modifications

Various methods have been developed so far for detecting N ⁶-methyladenosine (m⁶A). The total m⁶A level or the m⁶A status at individual positions on mRNA can be detected and quantified through some sequencing-independent biochemical methods, such as LC/MS, SCARLET, SELECT, and m⁶A-ELISA. However, the m⁶A-detection techniques relying on high-throughput sequencing have more effectively advanced the understanding about biological significance of m⁶A-containing mRNA and m⁶A pathway at a transcriptomic level over the past decade. Various SGS-based (Second Generation Sequencing-based) methods with different detection principles have been widely employed for this purpose. These principles include m⁶A-enrichment using antibodies, discrimination of m⁶A from unmodified A-base by nucleases, a fusion protein strategy relying on RNA-editing enzymes, and marking m⁶A with chemical/biochemical reactions. Recently, TGS-based (Third Generation Sequencing-based) methods have brought a new trend by direct m⁶A-detection. This review first gives a brief introduction of current knowledge about m⁶A biogenesis and function, and then comprehensively describes m⁶A-profiling strategies including their principles, procedures, and features. This will guide users to pick appropriate methods according to research goals, give insights for developing novel techniques in varying areas, and continue to expand our boundary of knowledge on m⁶A.