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

Abstract

Abstract 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.