Abstract
MicroRNAs (miRNAs) are a deeply conserved class of small, single stranded RNA molecules that post-transcriptionally regulate mRNA levels via several targeted degradation pathways. They are involved in a wide variety of biological processes and have been used to infer the deep evolutionary relationships of major groups such as the Metazoa. Here we have surveyed several adult tissues of the freshwater pulmonate Lymnaea stagnalis (the Great Pond Snail) for miRNAs. In addition we perform a shell regeneration assay to identify miRNAs that may be involved in regulating mRNAs directly involved in the shell-forming process. From seven mature tissues we identify a total of 370 unique precursor miRNAs that give rise to 336 unique mature miRNAs. While the majority of these appear to be evolutionarily novel, most of the 70 most highly expressed (which account for 99.8% of all reads) share sequence similarity with a miRBase or mirGeneDB2.0 entry. We also identify 10 miRNAs that are differentially regulated in mantle tissue that is actively regenerating shell material, 5 of which appear to be evolutionarily novel and none of which share similarity with any miRNA previously reported to regulate biomineralization in molluscs. One significantly down-regulated miRNA is predicted to target Lst-Dermatopontin, a previously characterized shell matrix protein from another freshwater gastropod. This survey provides a foundation for future studies that would seek to characterize the functional role of these molecules in biomineralization or other processes of interest.
Highlights
The diversity of proteins that animals employ to biomineralize is steadily being revealed, and it is clear that both deeply conserved and lineage specific elements play important roles in this process
A diverse clade of metazoans whose success is in part due to the evolutionary plasticity of the calcified shell, boast a diverse catalogue of shell-forming proteins that control all aspects of the biomineralization process from the initiation of mineral deposition, to regulation of the CaCO3 polymorph and inhibition of crystal growth
Our preliminary survey of the miRNA synthesis machinery present in a master transcriptome dataset of L. stagnalis returned sequences with significant similarity to all 30 query sequences (Additional file 2: Table 2). This suggests that all of the components necessary for the generation of miRNAs are present in the L. stagnalis genome and are actively expressed
Summary
The diversity of proteins that animals employ to biomineralize is steadily being revealed, and it is clear that both deeply conserved and lineage specific elements play important roles in this process. A diverse clade of metazoans whose success is in part due to the evolutionary plasticity of the calcified shell, boast a diverse catalogue of shell-forming proteins that control all aspects of the biomineralization process from the initiation of mineral deposition, to regulation of the CaCO3 polymorph and inhibition of crystal growth. All molluscs secrete shell-forming proteins (and other biomolecules such as polysaccharides and lipids) from the mantle tissue. These biomolecules are secreted either into the mantle cavity ( known as the pallial space, terms used to refer to the volume between the mantle tissue and the shell) or as a water-insoluble complex (the periostracum) that forms a matrix upon and within which calcification proceeds [8]
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