Osteoblasts orchestrate bone formation through the secretion of type I collagen and other constituents of the matrix on which hydroxyapatite crystals mineralize. Here, we show that TENT5A, whose mutations were found in congenital bone disease osteogenesis imperfecta patients, is a cytoplasmic poly(A) polymerase playing a crucial role in regulating bone mineralization. Direct RNA sequencing revealed that TENT5A is induced during osteoblast differentiation and polyadenylates mRNAs encoding Col1a1, Col1a2, and other secreted proteins involved in osteogenesis, increasing their expression. We postulate that TENT5A, possibly together with its paralog TENT5C, is responsible for the wave of cytoplasmic polyadenylation of mRNAs encoding secreted proteins occurring during bone mineralization. Importantly, the Tent5a knockout (KO) mouse line displays bone fragility and skeletal hypomineralization phenotype resulting from quantitative and qualitative collagen defects. Thus, we report a biologically relevant posttranscriptional regulator of collagen production and, more generally, bone formation.
Highly specialized enamel matrix proteins (EMPs) are predominantly expressed in odontogenic tissues and diverged from common ancestral gene. They are crucial for the maturation of enamel and its extreme complexity in multiple independent lineages. However, divergence of EMPs occured already before the true enamel evolved and their conservancy in toothless species suggests that non-canonical functions are still under natural selection. To elucidate this hypothesis, we carried out an unbiased, comprehensive phenotyping and employed data from the International Mouse Phenotyping Consortium to show functional pleiotropy of amelogenin, ameloblastin, amelotin, and enamelin, genes, i.e. in sensory function, skeletal morphology, cardiovascular function, metabolism, immune system screen, behavior, reproduction, and respiratory function. Mice in all KO mutant lines, i.e. amelogenin KO, ameloblastin KO, amelotin KO, and enamelin KO, as well as mice from the lineage with monomeric form of ameloblastin were affected in multiple physiological systems. Evolutionary conserved motifs and functional pleiotropy support the hypothesis of role of EMPs as general physiological regulators. These findings illustrate how their non-canonical function can still effect the fitness of modern species by an example of influence of amelogenin and ameloblastin on the bone physiology.
Enamel is the hardest tissue in mammalian organisms and is the layer covering the tooth. It consists of hydroxyapatite (HAP) crystallites, which mineralize on a protein scaffold known as the enamel matrix. Enamel matrix assembly is a very complex process mediated by enamel matrix proteins (EMPs). Altered HAP deposition or disintegration of the protein scaffold can cause enamel defects. Various methods have been established for enamel phenotyping, including MicroCT scanning with various resolutions from 9 μm for in vivo imaging to 1.5 μm for ex vivo imaging. With increasing resolution, we can see not only the enamel layer itself but also a detailed map of mineralization. To study enamel microstructure, we combine the MicroCT analysis with scanning electron microscopy (SEM), which enables us to perform element analyses such as calcium-carbon ratio. However, the methods mentioned above only show the result-already formed enamel. Stimulated emission depletion (STED) microscopy provides extra information about protein structure in the form of EMP localization and position before enamel mineralization. A combination of all these methods allows analyzing the same sample on multiple levels-starting with the live animal being scanned harmlessly and quickly, followed by sacrifice and high-resolution MicroCT scans requiring no special sample preparation. The biggest advantage is that samples remain in perfect condition for SEM or STED microscopic analysis.
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