Enhanced sarcoplasmic reticulum Ca(2+)-leak via ryanodine receptor type-2 (RyR2) contributes to the pathogenesis of atrial fibrillation (AF). Recent studies have shown that the level of RyR2 protein ...is elevated in atria of patients with paroxysmal AF, suggesting that microRNA-mediated post-transcriptional regulation of RyR2 might be an underlying mechanism. Bioinformatic analysis suggests that miR-106b and miR-93, members of the miR-106b-25 cluster, could bind to RyR2-3'-untranslated region and suppress its translation. Thus, we tested the hypothesis that loss of the miR-106b-25 cluster promotes AF via enhanced RyR2-mediated sarcoplasmic reticulum Ca(2+)-leak.
Quantitative real-time polymerase chain reaction showed that the levels of mature miR-106b, miR-93, and miR-25 were lower in atria of patients with paroxysmal AF when compared with patients in sinus rhythm. In vitro assay showed that miR-93 reduced RyR2-3'-untranslated region luciferase activity. Total RyR2 protein in atrial tissue of miR-106b-25(-/-) mice was increased by 42% when compared with wild-type littermates but still maintained a normal subcellular distribution. Ca(2+)-spark frequency and total sarcoplasmic reticulum Ca(2+)-leak were increased in atrial myocytes of miR-106b-25(-/-) mice. Telemetry ECG recordings revealed that miR-106b-25(-/-) mice exhibited more frequent atrial ectopy and were also more susceptible to pacing-induced AF than wild-type littermates. Increased sarcoplasmic reticulum Ca(2+)-release and AF susceptibility in miR-106b-25(-/-) mice were abolished by the RyR2 blocker K201.
These results suggest that miR-106b-25 cluster-mediated post-transcriptional regulation of RyR2 is a potential molecular mechanism involved in paroxysmal AF pathogenesis. As such, the miR-106b-25 cluster could be a novel gene-therapy target in AF associated with enhanced RyR2 expression.
The importance of translational regulation in tumour biology is increasingly appreciated. Here, we leverage polyribosomal profiling to prospectively define translational regulatory programs ...underlying epithelial-to-mesenchymal transition (EMT) in breast epithelial cells. We identify a group of ten translationally regulated drivers of EMT sharing a common GU-rich cis-element within the 3'-untranslated region (3'-UTR) of their mRNA. These cis-elements, necessary for the regulatory activity imparted by these 3'-UTRs, are directly bound by the CELF1 protein, which itself is regulated post-translationally during the EMT program. CELF1 is necessary and sufficient for both mesenchymal transition and metastatic colonization, and CELF1 protein, but not mRNA, is significantly overexpressed in human breast cancer tissues. Our data present an 11-component genetic pathway, invisible to transcriptional profiling approaches, in which the CELF1 protein functions as a central node controlling translational activation of genes driving EMT and ultimately tumour progression.
Scope
The uptake of dietary plant small RNAs (sRNAs) in consumers remains controversial, which is mainly due to low dietary content in combination with poor fractional absorption. MIR2911, among all ...the plant sRNAs including microRNAs, has been shown to be one of the most robustly absorbed sRNAs. Here we analyze the unusual abundance and unique genesis of MIR2911 during vegetable processing.
Methods and results
Using qRT‐PCR, the abundance of MIR2911 increased dramatically in macerated tissues while other microRNAs degraded. The accumulation of MIR2911 correlated with the degradation of the rRNAs, consistent with MIR2911 being derived from the 26S rRNA. Bioinformatic analysis predicts a microRNA‐like precursor structure for MIR2911; however, no reciprocal increase in the putative star‐strand was noted, and using an Arabidopsis mutation deficient in miRNA processing the accumulation of MIR2911 appeared Dicer independent. MIR2911 was incorporated into the mammalian RNA‐induced silencing complex as demonstrated in HEK293T cells, where transfected synthetic MIR2911 modestly suppressed the activity of a cognate luciferase reporter.
Conclusion
The genesis and amplification of MIR2911 post‐harvest is atypical, as traditional plant bioactives are less plentiful as vegetables lose freshness. These findings offer an explanation to the disparity in serum detection between MIR2911 and canonical plant‐based miRNAs.
The bioavailability of MIR2911, a particular plant small RNA (sRNA) derived from the 26S ribosomal RNA (rRNA), is higher when the plant food has been degraded. Increased amounts of MIR2911 correlated with the degradation of the rRNAs post harvest. This dietary sRNA may be like wine and cheese–getting better with age.
Mol. Nutr. Food Res. 2017, 61, 1600974.
DOI: 10.1002/mnfr.201600974
When vegetables such as cabbage are processed the small RNA MIR2911 (depicted as a hairpin), derived from the 26S rRNA, greatly ...multiply. The propagation of MIR2911 correlates with the degradation of the rRNA during processing. The enhancement of bioavailability post‐harvest makes this dietary RNA unique among putative plant bioactive compounds.
Myoblasts are mononucleated precursors of myofibers; they persist in mature skeletal muscles for growth and regeneration post injury. During myotonic dystrophy type 1 (DM1), a complex ...autosomal-dominant neuromuscular disease, the differentiation of skeletal myoblasts into functional myotubes is impaired, resulting in muscle wasting and weakness. The mechanisms leading to this altered differentiation are not fully understood. Here, we demonstrate that the calcium- and voltage-dependent potassium channel, KCa1.1 (BK, Slo1, KCNMA1), regulates myoblast proliferation, migration, and fusion. We also show a loss of plasma membrane expression of the pore-forming α subunit of KCa1.1 in DM1 myoblasts. Inhibiting the function of KCa1.1 in healthy myoblasts induced an increase in cytosolic calcium levels and altered nuclear factor kappa B (NFκB) levels without affecting cell survival. In these normal cells, KCa1.1 block resulted in enhanced proliferation and decreased matrix metalloproteinase secretion, migration, and myotube fusion, phenotypes all observed in DM1 myoblasts and associated with disease pathogenesis. In contrast, introducing functional KCa1.1 α-subunits into DM1 myoblasts normalized their proliferation and rescued expression of the late myogenic marker Mef2. Our results identify KCa1.1 channels as crucial regulators of skeletal myogenesis and suggest these channels as novel therapeutic targets in DM1.
Regulation of messenger ribonucleic acid (mRNA) subcellular localization, stability and translation is a central aspect of gene expression. Much of this control is mediated via recognition of mRNA 3' ...untranslated regions (UTRs) by microRNAs (miRNAs) and RNA-binding proteins. The gold standard approach to assess the regulation imparted by a transcript's 3' UTR is to fuse the UTR to a reporter coding sequence and assess the relative expression of this reporter as compared to a control. Yet, transient transfection approaches or the use of highly active viral promoter elements may overwhelm a cell's post-transcriptional regulatory machinery in this context. To circumvent this issue, we have developed and validated a novel, scalable piggyBac-based vector for analysis of 3' UTR-mediated regulation in vitro and in vivo. The vector delivers three independent transcription units to the target genome--a selection cassette, a turboGFP control reporter and an experimental reporter expressed under the control of a 3' UTR of interest. The pBUTR (piggyBac-based 3' UnTranslated Region reporter) vector performs robustly as a siRNA/miRNA sensor, in established in vitro models of post-transcriptional regulation, and in both arrayed and pooled screening approaches. The vector is robustly expressed as a transgene during murine embryogenesis, highlighting its potential usefulness for revealing post-transcriptional regulation in an in vivo setting.
The prevailing model of microRNA function is that the "seed region" (nt 2-8) is sufficient to mediate target recognition and repression. However, numerous recent studies have challenged this model, ...either by demonstrating extensive 3' pairing between physically defined miRNA-mRNA pairs or by showing in
that disrupted 3' pairing can result in impaired function in vivo. To test the importance of miRNA 3' pairing in a mammalian system in vivo, we engineered a mutant murine
allele in which the 5' half of the mature microRNA retains its wild-type sequence, but the 3' half's sequence has been altered to robustly disrupt predicted pairing to this latter region. Mice homozygous or hemizygous for this mutant allele are phenotypically indistinguishable from wild-type controls and do not recapitulate any of the immunopathology previously described for
-null mice. Our results indicate that 3' pairing is dispensable for the established myeloid function of this key mammalian microRNA.
Nitric oxide has been found to be an important biomessenger and bioregulator in the nervous, immune and cardiovascular systems. Since it carries such important roles in an organism, understanding the ...regulation of NO is important. While the mechanism of constitutive nitric oxide synthases has been widely understood, the regulation of iNOS has been unclear. There are several attempts trying to model the iNOS holoenzyme-CaM complex using several approaches such as kinetic, proteolysis, mass spectroscopy or circular dichroism. The previous studies provide valuable results including partial binding sites of NOS/CaM complex and the orientation of these two molecules. The ultimate goal of this project is to identify the binding sites on iNOS and CaM including intramolecular and intermolecular binding sites, and to combine the results from these experiments and from previous studies to simulate the binding pattern between iNOS and CaM. However, due to the difficulty of acquiring the sufficient amount of purified iNOS/CaM complex to achieve that goal. The purification of the iNOS/CaM complex from 4L BL21 (DE3) culture had been performed on 2', 5' –ADP Sepharose, Ni2+ NTA, and S200 gel filtration column. The purified active iNOS had been collected from only Ni2+ NTA column with 89% total iNOS yield and 200-fold purification. However, S200 gel filtration is required to achieve the more purified iNOS/CaM. By up scaling 4L BL21 (DE3) to 8L, the amount of the purified iNOS from Ni2+ NTA will be enough to applied to S200 gel filtration column. The restriction digestion of iNOS/CaM confirmed the CaM gene insert on pCACYC184 and confirmed 5' NdeI restriction site but did not confirm 3' XbaI site.
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