Mechanistic Insights into Amelogenin Alternative Splicing and Its Multifunctional Regulatory Roles: Implications for Amelogenesis Imperfecta and Signaling Pathways

/Abstract Amelogenin, a gene essential for dental enamel development, undergoes alternative splicing to produce different versions of its message, each leading to a unique amelogenin protein with distinct roles. A key result of this splicing occurs when a small gene segment, exon4, is removed. This process not only creates the primary protein needed for enamel formation but also generates a regulatory molecule called miR-exon4, a microRNA that may help refine enamel development. Mutations in the amelogenin gene cause X-linked Amelogenesis Imperfecta (X-AI), a genetic disorder characterized by weak or low-volume enamel. It is known that over 60% of disease-causing mutations do not directly alter the protein coding; instead, they disrupt the splicing process. However, researchers have only recently linked these mutations to the splicing of amelogenin. About one-quarter of these mutations occur specifically in exons 4 and 5. Our previous research has shown that these mutations affect exon4 splicing and miR-exon 4 production in cell models, suggesting a direct link to the disease. We found that these mutations happen in parts of the amelogenin gene recognized by splicing regulatory proteins, known as splicing factors. During alternative splicing, these proteins determine whether exon4 is included or skipped in the mRNA, which serves as the template for the final protein. Our preliminary study also showed that miR-exon4 can affect the expression of these splicing factors and is directly involved in the exon4 splicing process, creating a feedback loop that helps maintain normal enamel development. This proposal aims to investigate how exon4 splicing is regulated in both healthy and disease conditions, the specific role miR-exon4 plays in this regulation, and how this mechanism affects enamel formation. Our findings will reveal, for the first time, how exon4 splicing helps coordinate the various functions of amelogenin during enamel development. We will also examine how this process is disrupted in X-AI and how it links to key signaling pathways involved in producing healthy enamel. Since enamel is vital for protecting teeth and supporting normal function, issues in enamel formation can significantly impact an individual’s oral health and quality of life. Alternative splicing is emerging as a promising new avenue for drug development. Additionally, microRNAs— small molecules that help regulate gene activity—are already being investigated for medical therapies. This research integrates these two concepts. Our long-term goal is to understand the molecular mechanisms of enamel formation and to use this knowledge to develop innovative treatments for enamel defects. Ultimately, this work could lead to new therapies that correct faulty gene processing in patients with X-AI, providing a targeted approach to treating this currently untreatable condition. Project Number: 1R01DE035194-01A1 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of Dental and Craniofacial Research (NIDCR) | Principal Investigator: Yukiko Nakano | Institution: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO, SAN FRANCISCO, CA | Award Amount: $693,185 | Activity Code: R01 | Study Section: Special Emphasis Panel[ZRG1 MSOS-K (90)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11368408