Mapping multiple RNA modifications simultaneously by proximity barcode sequencing

RNA is subject to a multitude of different chemical modifications that collectively represent the epitranscriptome. Individual RNA modifications including N6-methyladenosine (m⁶A) on mRNA play essential roles in the posttranscriptional control of gene expression. Recent technological advances have enabled the transcriptome-wide mapping of certain RNA modifications, to reveal their broad relevance and characteristic distribution patterns. However, convenient methods that enable the simultaneous mapping of multiple different RNA marks within the same sample are generally lacking. Here we present EpiPlex RNA modification profiling, a bead-based proximity barcoding assay with sequencing readout that expands the scope of molecular recognition-based RNA modification detection to multiple targets, while providing relative quantification and enabling low RNA input. Measuring signal intensity against spike-in controls provides relative quantification, indicative of the RNA mod abundance at each locus. We report on changes in the modification status of HEK293T cells upon treatment with pharmacological inhibitors separately targeting METTL3, the dominant m⁶A writer enzyme, and the EIF4A3 component of the exon junction complex (EJC). The treatments resulted in decreased or increased m⁶A levels, respectively, without effect on inosine levels. Inhibiting the helicase activity of EIF4A3 and EIF4A3 knockdown both cause a significant increase of m⁶A sites near exon junctions, consistent with the previously reported role of EIF4A3 in shaping the m⁶A landscape. Thus, EpiPlex offers a reliable and convenient method for simultaneous mapping of multiple RNA modifications to facilitate epitranscriptome studies.