From m⁶A to Cap-Adjacent m⁶Am and Their Effects on mRNAs

Although RNA modifications were discovered decades ago, the identification of enzymes that write, read, and erase RNA modifications enabled their functional study and spawned the field of epitranscriptomics. Coupling that knowledge to new methods has enabled the precise pinpointing of epitranscriptomic modifications across the transcriptome plus the elucidation of their functional consequences. PCIF1 (Phosphorylated CTD Interacting Factor 1) was shown to add N6, 2’-O-dimethyladenosine (m⁶Am) marks at the first nucleotide after the 5’ N7-methylguanosine (m⁷G) cap. In this review, we discuss the epitranscriptomic regulation of mRNA in general, and focus on m⁷G cap-adjacent m⁶Am in particular. m⁶Am positions can now be distinguished from N6-methyladenosine (m⁶A) using new techniques leveraging PCIF1-knockout cells. Although m⁶Am modification sites can be detected precisely, conflicting data have been published regarding how cap-adjacent m⁶Am marks affect their host mRNA. Discrepancies in the data mean that the effects of cap-adjacent m⁶Am on mRNA stability, decapping, and translation continue to be debated. Finally, while PCIF1 is predominantly nuclear, a subset of results suggest a possible cytoplasmic role as well. Taken together, these contradictory results which employed different methodologies and cell lines means that further experiments are required to determine the ultimate biological function(s) of m⁷G cap-adjacent m⁶Am.