Extended Data Fig. 6: m⁶A loss leads to slight preference for proximal poly(A) site selection, but does not substantially shorten mRNAs.

a, m⁶A correlates with a slight preference for distal polyadenylation site (PAS) usage. DaPars⁴⁰ provides a percentage that predicts proximal (negative values) or distal (positive values) shifts in PAS usage relative to another condition. Each dot represents a unique transcript isoform. Best-fit line (blue) and standard deviation (light gray). P-values: Fisher’s exact test comparing control to Mettl3 KO transcripts. Benjamini-Hochberg adjustment (false discovery rate=5%) applied to all p-values before log transformation. Transcripts are assigned to the ‘proximal’ (6388 transcripts) and ‘distal’ (8425 transcripts) group if the change in site usage (that is, the relative use of proximal vs. distal PAS for all the transcript copies from a gene) is >5% in control relative to Mettl3 KO. Transcripts with <5% change are not assigned (1780 transcripts). b,c, We wanted to determine how overall length of transcripts could be affected by selection of distal or proximal PAS. Boxplot (b) depicts the length change between proximal and distal PAS usage in all transcripts (n = 16593) depicted in a. Boxplot depicts the median (heavy line), upper and lower quartile medians (box), and interquartile range (whiskers, IQR = 1.5). Outliers plotted separately. The median length change is ~272 nt. ~70% of alternative PAS will cause a length change of <0.5 kb. ~16% have an alternative PAS that will lead to a change in transcript length >1 kb. d, Proximal or distal PAS preferences do not substantially alter the length-dependent effect of mRNA enrichment in control stress granules. Longest transcript isoform is plotted on the y-axis. The effect is quantified for both ‘proximal APA’ transcripts (1960 transcripts) and ‘distal APA’ transcripts (2612 transcripts) as determined in a. Length-dependent enrichment is seen irrespective of changes in APA sites. e, Proximal or distal polyadenylation site preferences do not substantially alter the length-dependent effect of mRNA enrichment in Mettl3 KO SG. Analysis is performed as in d, except lengths are plotted against the enrichment of mRNAs in Mettl3 KO stress granules. In contrast to d, no clear relationship can be observed between SG enrichment for transcripts that show either distal or proximal APA site shifts in control cells. The fit line was measured using the correlation test (proximal, r = 0.14, p < 4.8×10^-10; distal, r = -0.07, p < 7.7×10^-).