Fig. 2. Pre-mRNA splicing suppresses m⁶A methylation in average-length exons.

(A and B) Left: schematic of specified BG CRY1 constructs. Blue regions indicate sequences derived from the CRY1 endogenous sequence, gray regions indicate sequences derived from rabbit beta-globin (BG). Number following CRY1 refers to the number of nucleotides of exonic sequence surrounding the CRY1 suppressed m⁶A site in the CRY1 endogenous mRNA that was cloned into the BG construct. Grey dot in the blue region denotes the suppressed m⁶A site; the number at the left and right of the m⁶A site shows the distance (nt) between the m⁶A site and the 3′ and 5′ splice site, respectively; the number next to the TSS shows the distance (nt) between the m⁶A site and the promoter. Δ denotes deletion of the specified intron(s). Details of each construct are described in the supplementary method. Right: m⁶A enrichment at a CRY1 suppressed m⁶A site. Primers amplifying a 62 nt-fragment containing the CRY1 suppressed m⁶A site. m⁶A enrichment was calculated as IP/input normalized to m⁶A-marked Gaussia luciferase RNA spike-in IP/input. Mean ± SEM, two-tailed t-test, *P < 0.05; **P < 0.01, ***P < 0.001. Three biological replicates.