Department of Orofacial Sciences, School of Dentistry, University of California, San Francisco, CA, USA.
Oral and Craniofacial Science, Graduate Division, University of California, San Francisco, CA, USA.
J Dent Res. 2023 Oct;102(11):1210-1219. doi: 10.1177/00220345231180572. Epub 2023 Aug 10.
Amelogenin plays a crucial role in tooth enamel formation, and mutations on X-chromosomal amelogenin cause X-linked amelogenesis imperfecta (AI). Amelogenin pre-messenger RNA (mRNA) is highly alternatively spliced, and during alternative splicing, exon4 is mostly skipped, leading to the formation of a microRNA (miR-exon4) that has been suggested to function in enamel and bone formation. While delivering the functional variation of amelogenin proteins, alternative splicing of exon4 is the decisive first step to producing miR-exon4. However, the factors that regulate the splicing of exon4 are not well understood. This study aimed to investigate the association between known mutations in exon4 and exon5 of X chromosome amelogenin that causes X-linked AI, the splicing of exon4, and miR-exon4 formation. Our results showed mutations in exon4 and exon5 of the amelogenin gene, including c.120T>C, c.152C>T, c.155C>G, and c.155delC, significantly affected the splicing of exon4 and subsequent miR-exon4 production. Using an amelogenin minigene transfected in HEK-293 cells, we observed increased inclusion of exon4 in amelogenin mRNA and reduced miR-exon4 production with these mutations. In silico analysis predicted that Ser/Arg-rich RNA splicing factor (SRSF) 2 and SRSF5 were the regulatory factors for exon4 and exon5 splicing, respectively. Electrophoretic mobility shift assay confirmed that SRSF2 binds to exon4 and SRSF5 binds to exon5, and mutations in each exon can alter SRSF binding. Transfection of the amelogenin minigene to LS8 ameloblastic cells suppressed expression of the known miR-exon4 direct targets, and , related to multiple pathways. Given the mutations on the minigene, the expression of has been significantly upregulated with c.155C>G and c.155delC mutations. Together, we confirmed that exon4 splicing is critical for miR-exon4 production, and mutations causing X-linked AI in exon4 and exon5 significantly affect exon4 splicing and following miR-exon4 production. The change in miR-exon4 would be an additional etiology of enamel defects seen in some X-linked AI.
成釉蛋白在牙釉质形成中起着至关重要的作用，X 染色体上的成釉蛋白基因突变会导致 X 连锁型釉质不全（AI）。成釉蛋白前信使 RNA（mRNA）高度可变剪接，在可变剪接过程中，外显子 4 大部分被跳过，导致形成 microRNA（miR-exon4），该 microRNA 被认为在牙釉质和骨骼形成中发挥作用。虽然可变剪接产生了具有功能的成釉蛋白变异体，但外显子 4 的剪接是产生 miR-exon4 的决定性的第一步。然而，调节外显子 4 剪接的因素尚不清楚。本研究旨在探讨导致 X 连锁 AI 的 X 染色体成釉蛋白外显子 4 和外显子 5 中的已知突变与外显子 4 的剪接和 miR-exon4 形成之间的关系。我们的结果显示，成釉蛋白基因外显子 4 和外显子 5 的突变，包括 c.120T>C、c.152C>T、c.155C>G 和 c.155delC，显著影响外显子 4 的剪接和随后的 miR-exon4 产生。使用转染 HEK-293 细胞的成釉蛋白小基因，我们观察到这些突变导致成釉蛋白 mRNA 中外显子 4 的内含增加，miR-exon4 的产生减少。计算机分析预测，富含丝氨酸/精氨酸的 RNA 剪接因子（SRSF）2 和 SRSF5 分别是外显子 4 和外显子 5 剪接的调节因子。电泳迁移率变动分析证实 SRSF2 结合外显子 4，SRSF5 结合外显子 5，每个外显子的突变都可以改变 SRSF 的结合。转染成釉蛋白小基因至 LS8 成釉细胞后，抑制了已知的 miR-exon4 直接靶基因的表达，这些基因与多个途径有关。鉴于小基因上的突变，c.155C>G 和 c.155delC 突变使 的表达显著上调。总之，我们证实外显子 4 的剪接对于 miR-exon4 的产生至关重要，外显子 4 和外显子 5 导致 X 连锁 AI 的突变显著影响外显子 4 的剪接和随后的 miR-exon4 产生。miR-exon4 的变化可能是一些 X 连锁 AI 中所见牙釉质缺陷的另一个病因。
