引起X连锁Ameloggenesis不全的突变改变Amelogenin外显子4的miRNA形成。

IF 8.3 2区 材料科学 Q1 MATERIALS SCIENCE, MULTIDISCIPLINARY
ACS Applied Materials & Interfaces Pub Date : 2023-10-01 Epub Date: 2023-08-10 DOI:10.1177/00220345231180572
R Shemirani, M H Le, Y Nakano
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引用次数: 0

摘要

釉蛋白在牙釉质形成中起着至关重要的作用,X染色体釉蛋白的突变导致X连锁釉形成不全(AI)。Amelogenin前信使RNA(mRNA)是高度选择性剪接的,在选择性剪接过程中,外显子4大多被跳过,导致微小RNA(miR-econ4)的形成,该微小RNA被认为在牙釉质和骨形成中发挥作用。在传递釉原蛋白的功能变化时,外显子4的选择性剪接是产生miR-econ4的决定性第一步。然而,调节外显子4剪接的因素尚不清楚。本研究旨在研究X染色体釉原蛋白第4外显子和第5外显子的已知突变(导致X连锁AI)、外显子4的剪接和miR-econ4的形成之间的关系。我们的结果显示,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,显著影响外显子的剪接和随后的miR-econ4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-econ4的产生。计算机分析预测富含Ser/Arg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。电泳迁移率变化分析证实SRSF2与外显子4结合,SRSF5与外显基因5结合,每个外显子的突变都可以改变SRSF的结合。将造釉素小基因转染到LS8成釉细胞中抑制了与多种途径相关的已知miR-econ4直接靶标Nfia和Prkch的表达。考虑到小基因上的突变,Prkch的表达随着c.155C>G和c.155delC突变而显著上调。总之,我们证实了外显子4剪接对miR-excon4的产生至关重要,并且在外显子和外显子5中引起X连锁AI的突变显著影响外显子的剪接和随后的miR-excon 4的产生。miR-econ4的变化可能是某些X连锁AI中牙釉质缺陷的另一个病因。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Mutations Causing X-Linked Amelogenesis Imperfecta Alter miRNA Formation from Amelogenin Exon4.

Mutations Causing X-Linked Amelogenesis Imperfecta Alter miRNA Formation from Amelogenin Exon4.

Mutations Causing X-Linked Amelogenesis Imperfecta Alter miRNA Formation from Amelogenin Exon4.

Mutations Causing X-Linked Amelogenesis Imperfecta Alter miRNA Formation from Amelogenin Exon4.

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.

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来源期刊
ACS Applied Materials & Interfaces
ACS Applied Materials & Interfaces 工程技术-材料科学:综合
CiteScore
16.00
自引率
6.30%
发文量
4978
审稿时长
1.8 months
期刊介绍: ACS Applied Materials & Interfaces is a leading interdisciplinary journal that brings together chemists, engineers, physicists, and biologists to explore the development and utilization of newly-discovered materials and interfacial processes for specific applications. Our journal has experienced remarkable growth since its establishment in 2009, both in terms of the number of articles published and the impact of the research showcased. We are proud to foster a truly global community, with the majority of published articles originating from outside the United States, reflecting the rapid growth of applied research worldwide.
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