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. 2015 Oct 1;29(19):2037–2053. doi: 10.1101/gad.269415.115

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© 2015 Ke et al.; Published by Cold Spring Harbor Laboratory Press

This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genesdev.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.

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Figure 4. — Higher m⁶A levels in the last exons correlates with more distal polyA site usage. (A) m⁶A is more abundant near proximal sites that are used less frequently (black line; left panel); when distal sites are used more, the m⁶A level is higher until the polyA site is reached (black line; right panel). The error bar indicates the standard error of the mean at each position. The shaded area highlights the major area of difference between two groups. (**) P < 10⁻³⁵, Fisher's exact test. “Used more“ means ≥60% usage of all polyA sites in the last exon, and “used less” means ≤40% usage. Other cutoffs, including 50% or 70%, produce the same findings. The expression of mRNAs in regions between proximal and distal polyA sites is adequate for m⁶A detection (RPKM ≥ 1). Mouse brain data are presented here; see also Supplemental Figure 5A for human data. (B) Distal APA sites in the last exons are used more often in brains for coexpressed mRNAs. APA site pairs with statistically significant differences in usage are highlighted in orange (higher usage of distal sites in the brain) or blue (higher usage of distal sites in the liver). FDR = 20%, Fisher's exact test. The same conclusion is true for FDR = 5% (Supplemental Fig. 5B). (C) m⁶A peak regions in the last exons have a higher enrichment value in the brain. “m⁶A peak enrich. value” is the m⁶A-IP reads normalized to the input reads for each m⁶A peak region. m⁶A peak regions with statistically significant differences in “m⁶A peak enrich. value” are highlighted in orange (higher in the brain) or blue (higher in the liver). FDR = 5%, Fisher's exact test. (D) Positional plot of m⁶A peaks around APA sites in the last exons. “m⁶A peak density (brain–liver)” for each position was calculated as the number of m⁶A peak regions that are significantly higher in the brain (orange points in C) in a 10-nt interval divided by the total number of mRNAs that contained this position. (Orange lines) More distal usage; (gray lines) no change in the brain. The expression of mRNAs in regions between the proximal and distal polyA sites is adequate for m⁶A detection in both tissues (RPKM ≥ 1). The error bar indicates the standard error of the mean. The shaded area highlights the major area of difference between two groups. (**) P < 10⁻²⁰, Fisher's exact test.