N⁶‐Methyladenosine mRNA modification regulates transcripts stability associated with cotton fiber elongation

N6 -Methyladenosine (m6 A) is the most abundant methylation modification in eukaryotic mRNA. The discovery of the dynamic and reversible regulatory mechanism of m6 A has greatly promoted the development of m6 A-led epitranscriptomics. However, the characterization of m6 A in cotton fiber is still unknown. Here, we reveal the potential link between m6 A modification and cotton fiber elongation by parallel m6 A-immunoprecipitation-sequencing (m6 A-seq) and RNA-seq analysis of fibers from the short fiber mutants Ligonliness-2 (Li2 ) and wild-type (WT). This study demonstrated a higher level of m6 A in the Li2 mutant, with the enrichment of m6 A modifications in the stop codon, 3'-untranslated region and coding sequence regions than in WT cotton. In the correlation analysis between genes containing differential m6 A modifications and differentially expressed genes, we identified several genes that could potentially regulate fiber elongation, including cytoskeleton, microtubule binding, cell wall and transcription factors (TFs). We further confirmed that the methylation of m6 A affected the mRNA stability of these fiber elongation-related genes including the TF GhMYB44, which showed the highest expression level in the RNA-seq data and m6 A methylation in the m6 A-seq data. Next, the overexpression of GhMYB44 reduces fiber elongation, whereas the silencing of GhMYB44 produces longer fibers. In summary, these results uncover that m6 A methylation regulated the expression of genes related to fiber development by affecting mRNA's stability, ultimately affecting cotton fiber elongation.