Extended Data Fig. 9: Alternative RPKM comparison of stress granule enrichment of mRNAs based on length.

a, Comparison of the percentage of reads per kilobase per million (RPKM) between stress granule and total mRNA samples grouped by transcript length. Average RPKM values for mRNAs containing m⁶A sites were calculated and compared across samples. The percentages are approximated to the last digit given. b, Percentage change between stress granule and total mRNA RPKM in control and Mettl3 KO grouped by mapped m⁶A sites. The data shows the percentage differences depicted between each of the categories presented in a. The percentages are approximated to the hundredth. In control MEFs, a length-dependent enrichment effect is observed for all mRNAs longer than 1.5 kb. In Mettl3 KO MEFs, only mRNAs that are very short ( < 1.5 kb) or very long ( > 5 kb) are enriched in stress granules. Notably, shorter mRNAs in the 0-1.5 kb group comprise 50-60% of total overall reads, indicating the abundance of these transcripts. These reads are greatly de-enriched in control stress granules. In contrast, these abundant, short transcripts are enriched in Mettl3 KO stress granules. c, Estimating the effects of m⁶A on the stress granule transcriptome. Blue arrowed lines depict observations in control MEFs, while red arrowed lines represent observations during in Mettl3 KO. In the left panel, the m⁶A-dependent effect on stress granule enrichment is shown. In cells with Mettl3, m⁶A-DF interactions exert a strong effect on mRNA recruitment. The m⁶A effect is absent in cells lacking Mettl3. In the right panel, the length effect on stress granule enrichment is shown. Under standard conditions, long RNAs interact with one another through RNA-RNA interactions and multivalent DF-m⁶A effects. In the absence of m⁶A, shorter mRNAs enter stress granules. Some longer mRNAs are affected to a lesser degree by loss of m⁶A owing to compensatory RNA-RNA or other RNA-protein interactions.