MicroRNA miR-378 Regulates Nephronectin Expression Modulating Osteoblast Differentiation by Targeting GalNT-7

MicroRNAs (miRNAs) are small fragments of single-stranded RNA containing 18-24 nucleotides, and are generated from endogenous transcripts. MicroRNAs function in post-transcriptional gene silencing by targeting the 3′-untranslated region (UTR) of mRNAs, resulting in translational repression. We have developed a system to study the role of miRNAs in cell differentiation. We have found that one of the miRNAs tested in our system (miR-378, also called miR-378*) plays a role in modulating nephronectin-mediated differentiation in the osteoblastic cell line, MC3T3-E1. Nephronectin is an extracellular matrix protein, and we have demonstrated that its over-expression enhanced osteoblast differentiation and bone nodule formation. Furthermore, we found that the nephronectin 3′-untranslated region (3′UTR) contains a binding site for miR-378. Stable transfection of MC3T3-E1 cells with miR-378 inhibited cell differentiation. MC3T3-E1 cells stably transfected with nephronectin exhibited higher rates of differentiation and nodule formation as compared with cells transfected with nephronectin containing the 3′UTR in the early stages of development, suggesting that endogenous miR-378 is present and active. However, in the later stages of MC3T3-E1 development, the differentiation rates were opposite, with higher rates of differentiation and nodule formation in the cells over-expressing the 3′UTR of nephronectin. This appeared to be the consequence of competition between nephronectin and UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 7 (GalNAc-T7 or GalNT7) for miR-378 binding, resulting in increased GalNT7 activity, which in turn lead to increased nephronectin glycosylation and product secretion, thereby resulting in a higher rate of osteoblast differentiation

Expression of Versican 3′-Untranslated Region Modulates Endogenous MicroRNA Functions

BACKGROUND: Mature microRNAs (miRNAs) are single-stranded RNAs that regulate post-transcriptional gene expression. In our previous study, we have shown that versican 3'UTR, a fragment of non-coding transcript, has the ability to antagonize miR-199a-3p function thereby regulating expression of the matrix proteins versican and fibronectin, and thus resulting in enhanced cell-cell adhesion and organ adhesion. However, the impact of this non-coding fragment on tumorigenesis is yet to be determined. METHODS AND FINDINGS: Using computational prediction confirmed with in vitro and in vivo experiments, we report that the expression of versican 3'UTR not only antagonizes miR-199a-3p but can also lower its steady state expression. We found that expression of versican 3'UTR in a mouse breast carcinoma cell line, 4T1, decreased miR-199a-3p levels. The decrease in miRNA activity consequently translated into differences in tumor growth. Computational analysis indicated that both miR-199a-3p and miR-144 targeted a cell cycle regulator, Rb1. In addition, miR-144 and miR-136, which have also been shown to interact with versican 3'UTR, was found to target PTEN. Expression of Rb1 and PTEN were up-regulated synergistically in vitro and in vivo, suggesting that the 3'UTR binds and modulates miRNA activities, freeing Rb1 and PTEN mRNAs for translation. In tumor formation assays, cells transfected with the 3'UTR formed smaller tumors compared with cells transfected with a control vector. CONCLUSION: Our results demonstrated that a 3'UTR fragment can be used to modulate miRNA functions. Our study also suggests that miRNAs in the cancer cells are more susceptible to degradation, due to its interaction with a non-coding 3'UTR. This non-coding component of mRNA may be used retrospectively to modulate miRNA activities

The T-Cell Receptor β Variable Gene Promoter Is Required for Efficient Vβ Rearrangement but Not Allelic Exclusion

To investigate the role of promoters in regulating variable gene rearrangement and allelic exclusion, we constructed mutant mice in which a 1.2-kb region of the Vβ13 promoter was either deleted (P13(−/−)) or replaced with the simian virus 40 minimal promoter plus five copies of Gal4 DNA sequences (P13(R/R)). In P13(−/−) mice, cleavage, rearrangement, and transcription of Vβ13, but not the flanking Vβ gene segments, were significantly inhibited. In P13(R/R) mice, inhibition of Vβ13 rearrangement was less severe and was not associated with any apparent reduction in Vβ13 cleavage. Expression of a T-cell receptor (TCR) transgene blocked cleavages at the normal Vβ13-recombination signal sequence junction and Vβ13 coding joint formation of both wild-type and mutant Vβ13 alleles. However, a low level of aberrant Vβ13 cleavage was consistently detected, especially in TCR transgenic P13(R/R) mice. These findings suggest that the variable gene promoter is required for promoting local recombination accessibility of the associated Vβ gene segment. Although the promoter is dispensable for allelic exclusion, it appears to suppress aberrant Vβ cleavages during allelic exclusion

Llgl1 Connects Cell Polarity with Cell-Cell Adhesion in Embryonic Neural Stem Cells.

Malformations of the cerebral cortex (MCCs) are devastating developmental disorders. We report here that mice with embryonic neural stem-cell-specific deletion of Llgl1 (Nestin-Cre/Llgl1(fl/fl)), a mammalian ortholog of the Drosophila cell polarity gene lgl, exhibit MCCs resembling severe periventricular heterotopia (PH). Immunohistochemical analyses and live cortical imaging of PH formation revealed that disruption of apical junctional complexes (AJCs) was responsible for PH in Nestin-Cre/Llgl1(fl/fl) brains. While it is well known that cell polarity proteins govern the formation of AJCs, the exact mechanisms remain unclear. We show that LLGL1 directly binds to and promotes internalization of N-cadherin, and N-cadherin/LLGL1 interaction is inhibited by atypical protein kinase C-mediated phosphorylation of LLGL1, restricting the accumulation of AJCs to the basolateral-apical boundary. Disruption of the N-cadherin-LLGL1 interaction during cortical development in vivo is sufficient for PH. These findings reveal a mechanism responsible for the physical and functional connection between cell polarity and cell-cell adhesion machineries in mammalian cells

Centimeter-Scale and Highly Crystalline Two-Dimensional Alcohol: Evidence for Graphenol (C6OH)

We report a chemical route to synthesize centimeter-scale stoichiometric "graphenol (C6OH1)", a 2D crystalline alcohol, via vapor phase hydroxylation of epitaxial graphene on Cu(111). Atomic resolution scanning tunneling microscopy revealed this highly-ordered configuration of graphenol and low energy electron diffraction studies on a large-area single crystal graphene film demonstrated the feasibility of the same superstructure being achieved at the centimeter length scale. Periodic density functional theory (DFT) calculations about the formation of C6(OH)(1) and its electronic structure are also reported

Comparative Study on Blowfly-Derived DNA and Camera Trapping in Assessing Mammalian Diversity in Subtropical Forests

Mammalian DNA derived from invertebrates (iDNA), including blowfly iDNA, is an alternative tool to conventional camera trapping in assessing mammalian diversity. The method has been used in tropical mammal surveillance but no attempt has been made to compare the efficacy of blowfly iDNA and camera trapping for monitoring mammal diversity in subtropical forests. We compared the blowfly iDNA monitoring with camera trapping to assess mammal diversity in the subtropical Jiulongfeng Nature Reserve (JLF), China, over a one-month period. The camera traps captured 2508 animal photos of 11 species belonging to four orders, eight genera, and eight families, whereas the blowfly iDNA method successfully detected ten species from six orders, eight genera, and eight families in JLF. Both methods were complementing each other instead of competing due to the low overlaps of mammal species detected. Of the total number of mammal species listed in JLF’s threatened list, 40% and 10% were detected through camera traps and blowfly iDNA methods, respectively. The estimated species richness curves indicated that combining camera traps and blowfly traps would increase the detection of mammal species. The strategy would significantly contribute to mammalian diversity surveillance and conservation programs in the tropical and subtropical forests