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pH has a great impact on the distribution, growth, behavior, and physiology in many aquatic animals. Here, we analyzed miRNA expression profiles of Chinese shrimp (Fenneropenaeus chinensis) from control pH (8.2) and low pH (6.5)-treated shrimp. Expression analysis identified 6 known miRNAs and 23 novel miRNAs with significantly different expression between control pH 8.2 and low pH 6.5; the predicted target genes of differentially expressed miRNAs were significantly enriched in organic acid metabolic process, oxidoreductase activity, coenzyme binding, cofactor binding, and collagen trimer. Moreover, target genes were significantly enriched in several Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways including citrate cycle, pyruvate metabolism, cytokine-cytokine receptor interaction, tight junction, carbon metabolism, etc. Our survey expanded the number of known shrimp miRNAs and provided comprehensive information about miRNA response to low pH stress. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12192-019-00989-x) contains supplementary material, which is available to authorized users.

Patients taking glucocorticoid or glucocorticoid-like drugs for an extended period of time may develop osteoporosis, termed glucocorticoid-induced osteoporosis (GIOP). GIOP is the most common form of secondary osteoporosis, but the mechanism underlying its development is unclear. In the present study, we used prednisolone to treat zebrafish larvae to investigate GIOP. Our RNA deep-sequencing (RNA-seq) results show that prednisolone affects genes known to act in the extracellular region. Therefore the extracellular region, extracellular matrix, and collagen trimer might be involved in glucocorticoid-induced osteoporosis. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that the focal adhesion signaling pathway is the most enriched signaling pathway in terms of differentially expressed genes (DEGs). In this pathway, integrin subunit alpha 10 (itga10) and integrin subunit beta like 1 (itgbl1), genes encoding two adapter proteins, were down-regulated in the prednisolone-treated larvae. Further experiments showed that prednisolone contributes to GIOP by down-regulating itga10 and itgbl1., Summary: Glucocorticoids may contribute to osteoporosis through effects on the extracellular region, the extracellular matrix and collagen trimer, and focal adhesion signaling pathway genes (itga10 and itgbl1).

Type I collagen is the most abundant protein in the human body and is composed of two α1(I) and one α2(I) polypeptides which assemble into a triple helix. For the proper assembly of the collagen triple helix, the individual polypeptides must be translated in coordination. Here, we show that serine-threonine kinase receptor-associated protein (STRAP) is tethered to collagen mRNAs by interaction with LARP6. LARP6 is a protein which directly binds the 5' stem-loop (5'SL) present in collagen α1(I) and α2(I) mRNAs, but it interacts with STRAP with its C-terminal domain, which is not involved in binding 5'SL. Being tethered to collagen mRNAs, STRAP prevents unrestricted translation, primarily that of collagen α2(I) mRNAs, by interacting with eukaryotic translation initiation factor 4A (eIF4A). In the absence of STRAP, more collagen α2(I) mRNA can be pulled down with eIF4A, and collagen α2(I) mRNA is unrestrictedly loaded onto the polysomes. This results in an imbalance of synthesis of α1(I) and α2(I) polypeptides, in hypermodifications of α1(I) polypeptide, and in inefficient assembly of the polypeptides into a collagen trimer and their secretion as monomers. These defects can be partially restored by supplementing STRAP. Thus, we discovered STRAP as a novel regulator of the coordinated translation of collagen mRNAs.

During peripheral nerve development, Schwann cells synthesize collagen type V molecules that contain alpha4(V) chains. This collagen subunit possesses an N-terminal domain (NTD) that contains a unique high affinity heparin binding site. The alpha4(V)-NTD is adhesive for Schwann cells and sensory neurons and is an excellent substrate for Schwann cell and axonal migration. Here we show that the alpha4(V)-NTD is released constitutively by Schwann cells both in culture and in vivo. In cultures of neonatal rat Schwann cells, alpha4(V)-NTD release is increased significantly by ascorbate treatment, which facilitates collagen post-translational modification and collagen trimer assembly. In peripheral nerve tissue, the alpha4(V)-NTD is localized to the region of the outer Schwann cell membrane and associated extracellular matrix. The released alpha4(V)-NTD binds to the cell surface and extracellular matrix heparan sulfate proteoglycans of Schwann cells. Pull-down assays and immunofluorescent staining showed that the major alpha4(V)-NTD-binding proteins are glypican-1 and perlecan. alpha4(V)-NTD binding occurs via a mechanism that requires the high affinity heparin binding site and that is blocked by soluble heparin, demonstrating that binding to proteoglycans is mediated by their heparan sulfate chains.

Collagen alpha1(I) mRNA is posttranscriptionally regulated in hepatic stellate cells (HSCs). Binding of protein factors to the evolutionary conserved stem-loop in the 5'-untranslated region (5' stem-loop) is required for a high level of expression in activated HSCs. The 5' stem-loop is also found in alpha2(I) and alpha1(III) mRNAs. Titration of the 5' stem-loop binding factors by a stably expressed RNA containing the 5' stem-loop (molecular decoy) may decrease the expression of these collagen mRNAs. We designed a 108-nt RNA that is transcribed from the optimized mouse U7 small nuclear RNA gene and contains the 5' stem-loop (p74WT decoy). This decoy accumulates in the nucleus and in the cytoplasm. When expressed in NIH 3T3 fibroblasts, the p74WT decoy decreased collagen alpha1(I) mRNA level by 60% and decreased collagen type I secreted into the cellular medium by 50%. We also expressed this decoy in quiescent rat HSCs by adenoviral gene transfer. Quiescent HSCs undergo activation in culture, resulting in a 60-70-fold increase in collagen alpha1(I) mRNA. The decoy decreases collagen alpha1(I) mRNA expression by 50-60% during activation of HSCs. It also decreases collagen alpha2(I) mRNA expression and collagen alpha1(III) mRNA expression. The cellular levels of collagen alpha1(I) propeptide and of disulfide-bonded collagen type I trimer are reduced by 70%. However, the p74WT decoy did not decrease alpha smooth muscle actin protein or the mRNA levels of glyceraldehyde-3-phosphate dehydrogenase and interleukin-6. The p74WT decoy was also introduced into activated human HSCs. In these cells, the decoy decreased collagen alpha1(I) propeptide and disulfide-bonded collagen trimer by 50-60%. These results indicate that the 5' stem-loop specifically regulates fibrillar collagen synthesis and represents a novel target for antifibrotic therapy. The molecular decoys provide a generalized method of assessing the functional significance of blocking the interactions of mRNA and proteins.

Collagen types IX, XII, and XIV are characterized by the presence of a highly conserved region comprising the most C-terminal triple helical domain (COL1, approximately 100 residues/chain) and 2 cysteines separated by 4 amino acid residues at the junction between this COL1 domain and the C-terminal non-triple helical domain (NC1). In order to better understand the functions of this conserved domain, we have constructed a recombinant minigene, comprising the sequence coding for an unrelated signal peptide and for the COL1 and NC1 domains of type XII collagen. This construct was placed under the control of the cytomegalovirus promoter and transfected into HeLa cells. The cells expressed the transfected minigene and the secreted chain, called alpha 1 (mini XII), could be detected by immunotransfer with an anti-peptide antibody recognizing an epitope found in the NC1 domain. Under conditions preventing the hydroxylation of prolyl residues (absence of ascorbate or presence of alpha alpha'-dipyridyl), interchain disulfide bridges did not form, while in the presence of ascorbate, disulfide-bonded (alpha 1 (mini XII))3 molecules were secreted. The collagenous nature and triple helical conformation of the trimeric molecule were ascertained by the differential resistances of the COL1 and NC1 domains to trypsin and collagenase digestions, respectively. Our data demonstrate that the NC1 and COL1 domains of type XII collagen contain the information necessary for trimer formation and that, contrary to the fibrillar collagen types, posttranslational modification of the triple helical domain is essential for assembly and disulfide bonding of the chains.

A factory needs a good distribution pipeline for its products, and so does a cell that manufactures collagen. Wilson et al. (1) describe a protein that helps pack several varieties of collagen into vesicles so they can be shipped from the cell. FOCAL POINT (Left to right) Khanhky Phamluong, Deanna Wilson, Andrew Peterson, Mark Solloway, and colleagues (not shown) took a closer look at how the protein Mia3 helps export collagen from cells. Compared with the endoplasmic reticulum in a cell from a normal mouse (center), the ER in a cell from a mouse lacking Mia3 is bloated (right). The researchers think that without Mia3’s assistance collagen piles up inside the ER. PHOTO COURTESY OF KHANHKY PHAMLUONG Collagen was one of the first proteins identified, but researchers are still trying to elucidate the mechanics of its secretion. After their synthesis in the ER, individual collagen molecules interweave to form a trimer (2) and then travel to the Golgi apparatus for further modification. One mystery is how a stiff collagen trimer that is more than 300 nm long could cram into the standard COPII-coated vesicles—which are only about 60 nm to 80 nm in diameter—that ferry cargo from the ER to the Golgi (3). Researchers surmise that cells deploy capacious vesicles that can accommodate collagen trimers, but they haven't spotted these carriers in vivo. A further mystery is what shepherds collagen into transport vesicles. A previous study (4) suggested that the protein Mia3 performs the job for collagen VII, one of the more than 20 varieties of the extracellular matrix protein. However, the work suggested that Mia3 doesn't load another version of the protein, collagen I. To find out more about Mia3’s function, Wilson et al. deleted the gene from mice. The animals died at birth, and their stunted skeleton and fragile skin pointed to faulty collagen secretion. Wilson et al. discovered that cartilage formation in the animals’ bones was tardy and that chondrocytes, cells specialized to synthesize cartilage, were slow to mature. As a result of these and other defects, the skeleton failed to mineralize. The consequences of losing Mia3 went beyond the skeleton. Collagen built up in cells throughout the animals’ bodies, the researchers found. Electron microscopy vividly portrayed the secretory stagnation. The ER in chondrocytes was bloated, presumably jam-packed with collagen. “Mia3 is clearly a facilitator of collagen processing in the ER.” Cells from the Mia3 knockout mice failed to export not only collagen VII but at least five other collagen varieties—including collagen I—that span several classes of the protein. The collagen buildup also spurred cells to activate the unfolded protein response, a stress reaction that kicks in when misfolded proteins accumulate in the ER. “Mia3 is clearly a facilitator of collagen processing in the ER,” says senior author Mark Solloway. The protein is in the right place and has the right structure to help bundle collagen into vesicles for shipment to the Golgi. Mia3 spans the ER membrane, and one end of the protein hooks onto collagen, whereas the other end attaches to the COPII coat of a cargo-ready vesicle. The work suggests that Mia3 is not a specialist and loads multiple collagen varieties. Several questions about Mia3 remain unanswered. The researchers found that secretion of two large extracellular matrix (ECM) proteins, aggrecan and fibronectin, was normal in the knockout mice. Whether Mia3 helps the ER ship other large proteins is uncertain. “The next hurdle will be to define the full repertoire of targets,” says Solloway. Another lingering question is whether the protein works alone. Cells from the knockout mice managed to extrude some collagen, suggesting that there may be additional collagen export mechanisms. The health impact of changes in Mia3 levels also remains unclear. Although studies have found that cells in several cancer types turn down Mia3 production, the protein's function in tumors—and whether that role involves collagen secretion—is murky. In addition, recent studies linked several polymorphisms in the Mia3 gene to an increased risk of heart attack. Collagen forms much of the cap of an atherosclerotic plaque. By reducing collagen secretion, these Mia3 variants might weaken the cap, causing it to fracture and trigger a heart attack. Alternatively, collagen-rich tissues, including the blood vessels, may be prone to cellular stress if secretion is impaired.

Structurally abnormal type I collagen was identified in the dermis, bone, and cultured fibroblasts obtained from a baby with lethal perinatal osteogenesis imperfecta. Two-dimensional gel electrophoresis of the CNBr peptides demonstrated that the alpha 1(I)CB7 peptide from the alpha 1(I)-chain of type I collagen existed in a normal form and a mutant form with a more basic charge distribution. This heterozygous peptide defect was not detected in the collagens from either parent. The defect was localized to a 224-residue region at the NH2 terminus of the alpha 1(I)CB7 peptide by mammalian collagenase digestion. Analysis of unhydroxylated collagens produced in cell culture indicated that the mutant alpha 1(I)CB7 migrated faster on electrophoresis suggesting that the abnormality may be a small deletion or a mutation that alters sodium dodecyl sulfate binding. The post-translational hydroxylation of lysine residues was increased in the CB7 peptide and also in peptides CB3 and CB8 which are toward the NH2 terminus of the alpha 1(I)-chain. The COOH-terminal CB6 peptide was normally hydroxylated. These findings support the proposal that the lysine overhydroxylation resulted from a perturbation of helix propagation from the COOH to NH2 terminus of the collagen trimer caused by the structural defect in alpha 1(I)CB7.