Figure 3. Depletion of PCIF1 distinguishes m6A and m6Am in transcriptome-wide 6mA maps.
A. Metagene of miCLIP reads in wild-type and PCIF1 knockout (KO) HEK293T cells. Shown is a metagene analysis of reads from the wild-type or PCIF1 KO miCLIP dataset. The first nucleotide of each read (with respect to the RNA strand) was extracted and plotted. Reads in the 5′UTR were lost in the PCIF1 knockout, suggesting a complete loss of m6Am in the PCIF1 knockout cells.
B. DREME motif search within called peaks show the DRACH motif as the most enriched in all datasets, consistent m6A as the most abundant 6mA-containing nucleotide mapped by miCLIP.
C. miCLIP peaks can be identified as m6Am or m6A based on their decrease in PCIF1 KO cells. Genome tracks were plotted for RPL35 and KDELR2 with called m6A sites (FDR<0.1) and m6Am sites indicated by red circles and blue triangle, respectively. Zoomed insets show m6Am peaks can be distinguished from nearby m6A sites.
D. The previously annotated m6Am site in RACK1 is actually a 5′UTR m6A. The TSS-proximal peak in RACK1 is not affected in the PCIF1 knockout and overlaps with a DRACH motif.
E. Metagene analysis of PCIF1 KO-validated m6Am sites shows m6Am sites throughout the 5′UTR and in the transcript body. Shown is a metagene of the exact sites of m6Am within the PCIF1-dependent peaks as determined by A to T transitions and the read drop-off method. The metagene reveals an overall enrichment at the TSS, with some sites that appear to be within the CDS and 3′UTR.
F. DREME motif search of the nucleotides surrounding each m6Am was performed confirms the previously reported BCA motif, and shows that the promoter sequence upstream of the m6Am is GC-enriched.
See also Figure S2.