A splicing-derived microRNA from amelogenin exon4 regulates enamel formation via control of exon4 splicing and amelogenin expression

Amelogenin, essential for enamel formation, is encoded by the alternatively spliced gene Amelx/AMELX, with exon4 splicing producing major protein isoforms and a microRNA (miR-exon4). We previously reported that miR-exon4 regulates osteoblast and ameloblast function via the Nfia- and Prkch-Runx2 axis and may be involved in X-linked Amelogenesis Imperfecta associated with exon4 splicing defects. This study investigates miR-exon4’s role in enamel defect development and exon4 splicing. A miR-exon4 mimic or inhibitor was applied to mouse pups during enamel development. One-week inhibition of miR-exon4 reduced enamel mineralization, shortened the early mineralization phase, and lowered RUNX2 expression, leading to increased Amelx mRNA and amelogenin protein levels. A 24-hour inhibition enhanced exon4 splicing without affecting overall Amelx expression, suggesting a direct role in splicing regulation. MiR-exon4 inhibition also altered the expression of Serine/Arginine-rich splicing factors (SRSFs), indicating indirect modulation of exon4 splicing. Nuclear localization of miR-exon4 was confirmed using an Amelx minigene, and a mutant form that reduced nuclear miR-exon4 levels impaired its binding to Amelx pre-mRNA. A binding site of miR-exon4 at the intron4 branch point further supports its direct involvement in exon4 splicing. These findings suggest a role for miR-exon4 in regulating both amelogenin expression and exon4 splicing, leading to enamel defects when absent.