Figure 4. Internally mapped m⁶Am sites reflect m⁶Am in mRNA isoforms with alternative TSSs.

A. A metaplot centered on the closest RefSeq TSS for each called m⁶Am site shows most m⁶Am sites are found downstream of the annotated start site, but not at the annotated start site. The proportion of m⁶Am directly at the annotated TSS, or up- or downstream is shown.

B. A metaplot analysis of m⁶Am locations using GENCODE TSS annotations shows higher overlap with TSSs. GENCODE annotations include more transcript isoforms and TSSs than RefSeq.

C. A metaplot of the distance from each m⁶Am site to the closest CAGE peak in the FANTOM5 database shows that m⁶Am sites are indeed TSSs. Here, the overlap of m⁶Am was highest, suggesting that m⁶Am sites are selectively localized to TSSs and not internal nucleotides within mRNA.

D. The m⁶Am mapping to the annotated 5′UTR of the YBX1 transcript reflects a transcript isoform. The PCIF1-dependent 6mA peak in YBX1 maps within the annotated 5′UTR of YBX1. However, this peak overlaps with a CAGE site (orange triangles), indicating the existence of a transcript isoform that initiates at this 6mA site. m⁶Am peaks that appear within the 5′UTR reflect m⁶Am in transcript isoforms with alternative TSSs. The exact m⁶Am site (blue triangle) was determined using the A to T transition within the PCIF1-dependent peak.

E. Most m⁶Am are found in the annotated 5′UTR of transcripts.

F. The internally mapping m⁶Am in YOD1 derives from a TSS of a YOD1 transcript isoform. The m⁶Am peak in YOD1 begins beyond the start codon of both annotated isoforms. CAGE peaks (orange triangles) suggest this is indeed a TSS.

G. A metaplot analysis of CDS and 3′UTR mapping m⁶Am sites show overlap with CAGE data, indicating that m⁶Am occurs at TSSs. The closest CAGE peak to each of the 6% of sites that appeared to not map to the 5′UTR (E) was calculated and plotted.

See also Figure S3.