Abstract
Biomineralization is a sophisticated biological process precisely regulated by multiple molecules and pathways. Accumulating miRNAs have been identified in invertebrates but their functions in biomineralization are poorly studied. Here, an oyster species-specific miRNA, novel_miR_1 was found to regulate biomineralization in Pinctada fucata. Target prediction showed that novel_miR_1 could target Prisilkin-39 and ACCBP by binding to their coding sequences (CDS). Tissue distribution analysis revealed that the expression level of novel_miR_1 was highest in the mantle, which was a key tissue participating in biomineralization. Gain-of-function assay in vivo showed that biomineralization-related genes including Prisilkin-39 and ACCBP were down-regulated and shell inner surfaces of both prismatic and nacreous layer were disrupted after the over-expression of novel_miR_1, indicating its dual roles in biomineralization. Furthermore, the shell notching results indicated that novel_miR_1 was involved in shell regeneration. Dual-luciferase reporter assay in vitro demonstrated that novel_miR_1 directly suppressed Prisilkin-39 and ACCBP genes by binding to the CDS regions. Taken together, these results suggest that novel_miR_1 is a direct negative regulator to Prisilkin-39 and ACCBP and plays an indispensable and important role in biomineralization in both prismatic and nacreous layer of P. fucata.
Highlights
Biomineralization is a process during which inorganic ions are orderly deposited under biological regulation of biomacromolecules[1,2]
It has been found that miRNAs play an important role in bone and teeth biomineralization in vertebrates[20,21]
Pinctada fucata (P. fucata) is an ideal model organism for studying biomineralization, from which researchers have identified a great number of SMPs7,8 including Prisilkin-3936 and ACCBP37. miRNAs involved in shell formation of this oyster should be given attention because they can play important roles by modulating the gene expression of shell matrix proteins (SMPs)
Summary
Biomineralization is a process during which inorganic ions are orderly deposited under biological regulation of biomacromolecules[1,2]. Based on the significance of miRNAs in regulation of gene expression, more research on the biomineralization-related miRNAs is needed to explore their roles in shell formation. MiRNAs involved in shell formation of this oyster should be given attention because they can play important roles by modulating the gene expression of SMPs. In previous studies, researchers have obtained a series of miRNAs in P. fucata by deep sequencing and computational prediction[26,27,38]. It was found that pm-miR-183 participated in nacre formation by targeting 3’UTR of PmRunt, which was a runt-like transcriptional factor involved in biomineralization possibly through promoting the expression of collagen VI-like and Nacrein[41]. There has been no report verifying a species-specific miRNA involved in both prismatic and nacreous layer formation by binding to the CDS regions
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