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.