The Exon 6ABC Region of Amelogenin mRNA Contribute to Increased Levels of Amelogenin mRNA through Amelogenin Protein-enhanced mRNA Stabilization

Liming Xu,Hidemitsu Harada,Akiyoshi Taniguchi

doi:10.1074/jbc.m605406200

Liming Xu, Hidemitsu Harada + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m605406200

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Abstract

We recently demonstrated that the reuptake of full-length amelogenin protein results in increased levels of amelogenin mRNA through enhanced mRNA stabilization (Xu, L., Harada, H., Tamaki, T. Y., Matsumoto, S., Tanaka, J., and Taniguchi, A. (2006) J. Biol. Chem. 281, 2257-2262). Here, we examined the molecular mechanism of enhanced amelogenin mRNA stabilization. To identify the cis-regulatory region within amelogenin mRNA, we tested various reporter systems using a deletion series of reporter plasmids. A deletion at exon 6ABC of amelogenin mRNA resulted in a 2.5-fold increase in the amelogenin mRNA expression level when compared with that of full-length mRNA, indicating that a cis-element exists in exon 6ABC of amelogenin mRNA. Furthermore, Northwestern analysis demonstrated that amelogenin protein binds directly to its mRNA in vitro, suggesting that amelogenin protein acts as a trans-acting protein that specifically binds to this cis-element. Moreover, recombinant mouse amelogenin protein extended the half-life of full-length amelogenin mRNA but did not significantly alter the half-life of exon 6ABC-deletion mutant mRNA. The splice products produced by deletion of exon 6ABC are known as leucine-rich amelogenin peptides and have signaling effects on cells. Our findings also suggest that the regulation of full-length amelogenin protein expression differs from the regulation of leucine-rich amelogenin peptide expression.

Highlights

Amelogenin is a major component of enamel matrix
We found that the exon 6ABC region of amelogenin mRNA is involved in amelogenin mRNA instability and amelogenin protein-mediated stability and that full-length amelogenin mRNA binds to amelogenin protein in vitro
These results suggest that the mRNA expression of LRAP, which results from the deletion of exon 6ABC, is not affected by amelogenin protein-mediated post-transcriptional regulation, which is different to full-length amelogenin mRNA

Summary

Introduction

Amelogenin is a major component of enamel matrix. Amelogenin is unique in its localization on both X-chromosomes and Y-chromosomes in cows, pigs, and humans and on the X-chromosomes in mice [1,2,3,4] rather than on chromosome 4q like other enamel- and mineralization-associated proteins. The smaller splice products, produced upon the deletion of exon 6ABC, are known as the leucine-rich amelogenin peptides or LRAPs.2 LRAPs have been shown to act differently, as signaling molecules affecting odontogenic and other cell types [12,13,14,15] The larger forms, those that contain the intact proline-rich, hydrophobic exon 6 domains, are important for enamel mineralization (for review, see Ref. 17). We found that the exon 6ABC region of amelogenin mRNA is involved in amelogenin mRNA instability and amelogenin protein-mediated stability and that full-length amelogenin mRNA binds to amelogenin protein in vitro These results suggest that the mRNA expression of LRAP, which results from the deletion of exon 6ABC, is not affected by amelogenin protein-mediated post-transcriptional regulation, which is different to full-length amelogenin mRNA. The regulation of full-length amelogenin protein expression differs from the regulation of LRAP expression

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