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

Abstract

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.

Keywords: RNA splicing factors; alternative splicing; ameloblasts; amelogenesis; dental enamel; developmental defects of enamel.

Figure 1.

Alternative splicing and exon splicing enhancers (ESEs). (A) Models for skipping or inclusion of an alternative exon. (Upper) In most cases, an alternative exon (E2) is skipped. It is because of the strong Ser/Arg-rich RNA splicing factor (SRSF) binding site on the conserved exons (E1 or E3). (Lower) The alternative exon is included only when the binding of the SRSF to the ESE on the conserved exon is disrupted or the binding of an SRSF to the weak ESE on the alternative exon is enhanced. (B) X-linked amelogenesis imperfecta causative mutations on exon4 and exon5 occur in putative ESE sequences for SRSF2 or SRSF5 binding, respectively. The mutation in exon4 is predicted to enhance SFSR2 binding (Cho et al. 2014), and the 4 mutations in exon5 are predicted to disable SRSF5 binding. Both of them, in turn, are expected to suppress exon4 splicing.

Figure 2.

Quantitative polymerase chain reaction analysis of messenger RNA (mRNA) including exon4 and miR-exon4. (A–D) Total amelogenin derived from minigenes was amplified using an exon2-PA28956 primer set. (E–H) Total amelogenin from entire HEK cells was amplified using an exon2-ex6d primer set. In both detection methods, total amelogenin amplification is not affected by the mutations on the minigene except c.155delC mutation (D and H). (I, K, M, O) Compared to wild-type (WT), all mutations result in significantly more inclusion of exon4 in amelogenin mRNA. (J, L, N, P) All mutations cause less production of mature miR-exon4 compared to the WT control. Data are plotted on the graph, and bars are shown as ± SD. Statistical analysis was done with an independent Student’s t test. **P < 0.01. *P < 0.05. ns, not significant.

Figure 3.

Exon4 inclusion in different forms of amelogenin messenger RNA (mRNA). (A) Alternative splicing variants of amelogenin mRNA derived from minigenes were detected by gel electrophoresis. The long form and short form of amelogenin (Amel) were determined according to the amplicon’s size. The pre–gel hybridization determined bands containing exon4 (red arrows) with an exon4 probe (Appendix Fig. 2). (B) In wild-type (WT) samples, the expression level of the long form of amelogenin containing exon4 is higher than the short form by comparing the quantitative polymerase chain reaction using F:exon4 and R:exon5/6d (for short form) and F:exon4 and R:exon6d (for long form) primer sets. (C–F) Compared to the WT, all mutations cause upregulation of exon4 inclusion in the long-form amelogenin mRNA. (G, I, J) c.120T>C, c.155C>G, and c.155delC mutations significantly upregulate the exon4 inclusion in the short form of amelogenin mRNA, while c.152C>T mutation slightly downregulates the exon4 inclusion (H). Data are plotted on the graph, and bars are shown as ± SD. Statistical analysis was done with an independent Student’s t test. *P < 0.05. **P < 0.01. ****P < 0.0001. ns, not significant.

Figure 4.

Ser/Arg-rich RNA splicing factors (SRSF) 2 and SRSF5 to regulate exon4 splicing. (A) Electrophoretic mobility shift assay (EMSA) of exon4 RNA and SRSF2. The presence of super-shifted bands by mixing exon4 wild-type (WT) RNA and SRSF2 and the disappearance of the super shift by further adding a competitor confirm that SRSF2 specifically bounds exon4 WT via 3 exon splicing enhancer (ESE) forming complexes. Possible complex formations with SRSF2 and RNA are shown in Appendix Figure 3. With c.120T>C mutation, complex 2 and 3 portions increase. (B) EMSA of exon5 RNA and SRSF5. The presence of super-shifted bands by mixing exon5 WT RNA and SRSF5 and the disappearance of the super shift by adding competitor confirm that SRSF5 bounds exon5 WT via 2 ESE forming complexes 1 and 2. Possible complex formations with SRSF5 and RNA are shown in Appendix Figure 4. With c.155C>G mutation, complex 1 shifted back to the free RNA. With c.155del mutation, complex 1 super-shifted to complex 2. No change in complex formation with c.152C>T mutation. (C) Srsf2 and Srsf5 expressions in mouse enamel organs at P0 (presecretory stage) and P5 (secretory stage). Data are plotted on the graph, and bars are shown as ± SD. Total animal number = 8. Statistical analysis was done with an independent Student’s t test. **P < 0.01. ***P < 0.001. (D) Immunostaining of SRSF2 and SRSF5 on presecretory and secretory ameloblasts (Am) of mandibular incisor. The immunoreaction is in red. The counterstaining is in light green. Bar: 20 µm. (E) Proposed process for exon4 splicing and formation of mature miR-exon4. When bound to ESEs, the SRSFs recruit small nuclear RNAs (U1–6) to form the spliceosome complex (Auyeung et al. 2013) and define exons or introns. When SRSF5 binds to exon5 in pre-mRNA, exon4 is skipped. The spliced introns and exon4 are further processed to be a mature miRNA. When the binding of SRSF5 to exon5 is weakened, or the binding of SRSF2 to exon4 is enhanced, exon4 is included in mRNA.

Figure 5.

Expression of miR-exon4 direct targets in LS8 cells. (A) Transfecting wild-type (WT) minigene to LS8 cells significantly reduces the expression of Prkch. (B) The c.155C>G and c.155delC mutations upregulate the Prkch expression, reversing the effect of minigene WT in the LS8 cells. (C) WT minigene suppresses Nfia expression. (D) None of the mutations further alter Nfia expression caused by the WT minigene. Data are plotted on the graph, and bars are shown as ± SD. Statistical analysis was done with an independent Student’s t test (A and C) or multiple t tests with Bonferroni correction following 1-way analysis of variance (B, D). **P < 0.01. ***P < 0.001. ns, not significant.