Mutations Causing X-Linked Amelogenesis Imperfecta Alter miRNA Formation from Amelogenin Exon4.
Mutations Causing X-Linked Amelogenesis Imperfecta Alter miRNA Formation from Amelogenin Exon4.
复制标题
DOI：
10.1177/00220345231180572
复制
发表时间：
2023-10
影响因子：
7.6
通讯作者：
中科院分区：
文献类型：
--
作者：
釉原蛋白在牙釉质形成中起着至关重要的作用，X染色体上的釉原蛋白突变导致X连锁的釉质发生突变（AI）。釉原蛋白前信使RNA（mRNA）是高度可变剪接的，并且在可变剪接过程中，外显子4大部分被跳过，导致已经被认为在釉质和骨形成中起作用的microRNA（miR-exon 4）的形成。在递送釉原蛋白蛋白的功能变异的同时，外显子4的选择性剪接是产生miR-exon 4的决定性的第一步。然而，调节外显子4剪接的因素还不清楚。本研究旨在探讨X染色体釉原蛋白外显子4和外显子5中已知的导致X连锁AI的突变、外显子4的剪接和miR-exon 4形成之间的关联。我们的研究结果显示釉原蛋白基因外显子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-exon 4的产生。使用在HEK-293细胞中转染的釉原蛋白小基因，我们观察到釉原蛋白mRNA中外显子4的包含增加，并且这些突变减少了miR-exon 4的产生。计算机模拟分析预测富含Ser/Arg的RNA剪接因子（SRSF）2和SRSF 5分别是外显子4和外显子5剪接的调控因子。电泳迁移率变动分析证实SRSF 2与外显子4结合，SRSF 5与外显子5结合，并且每个外显子中的突变可以改变SRSF结合。将釉原蛋白小基因转染到LS 8成釉细胞中抑制了已知的miR-外显子4直接靶点Nfia和Prkch的表达，这些靶点与多种途径有关。考虑到小基因上的突变，Prkch的表达已经用c.155C>G和c.155delC突变显著上调。总之，我们证实了外显子4剪接对miR-exon 4的产生至关重要，并且在外显子4和外显子5中引起X连锁AI的突变显著影响外显子4剪接和随后的miR-exon 4产生。miR-exon 4的变化可能是某些X连锁AI中所见釉质缺损的另一病因。
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, Nfia and Prkch, related to multiple pathways. Given the mutations on the minigene, the expression of Prkch 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.
登录
查看更多内容
影响因子：
14.9
作者：
Cartegni, L;Wang, JH;Krainer, AR
通讯作者：
Krainer, AR
影响因子：
3
作者：
CHEN, LS;COUWENHOVEN, RI;SNEAD, ML
通讯作者：
SNEAD, ML
影响因子：
7.6
作者：
Kida, M.;Sakiyama, Y.;Ariga, T.
通讯作者：
Ariga, T.
影响因子：
1.9
作者：
Lacerda-Pinheiro, Sally;Jegat, Nadege;Poliard, Anne
通讯作者：
Poliard, Anne
影响因子：
2.7
作者：
Michon, Frederic;Tummers, Mark;Thesleff, Irma
通讯作者：
Thesleff, Irma