Gene regulation by translational inhibition is determined by Dicer partnering proteins

MicroRNAs (miRNAs) are small regulatory RNAs produced by Dicer proteins that regulate gene expression in development and adaptive responses to the environment1,​2,​3,​4. In animals, the degree of base pairing between a miRNA and its target messenger RNA seems to determine whether the regulation occurs through cleavage or translation inhibition1. In contrast, the selection of regulatory mechanisms is independent of the degree of mismatch between a plant miRNA and its target transcript5. However, the components and mechanism(s) that determine whether a plant miRNA ultimately regulates its targets by guiding cleavage or translational inhibition are unknown6. Here we show that the form of regulatory action directed by a plant miRNA is determined by DRB2, a DICER-LIKE1 (DCL1) partnering protein. The dependence of DCL1 on DRB1 for miRNA biogenesis is well characterized7,​8,​9, but we show that it is only required for miRNA-guided transcript cleavage. We found that DRB2 determines miRNA-guided translational inhibition and represses DRB1 expression, thereby allowing the active selection of miRNA regulatory action. Furthermore, our results reveal that the core silencing proteins ARGONAUTE1 (AGO1) and SERRATE (SE) are highly regulated by miRNA-guided translational inhibition. DRB2 has been remarkably conserved throughout plant evolution, raising the possibility that translational repression is the ancient form of miRNA-directed gene regulation in plants, and that Dicer partnering proteins, such as human TRBP, might play a similar role in other eukaryotic systems.

Translational Up-Regulation and High-Level Protein Expression from Plasmid Vectors by mTOR Activation via Different Pathways in PC3 and 293T Cells

Background: Though 293T cells are widely used for expression of proteins from transfected plasmid vectors, the molecular basis for the high-level expression is yet to be understood. We recently identified the prostate carcinoma cell line PC3 to be as efficient as 293T in protein expression. This study was undertaken to decipher the molecular basis of high-level expression in these two cell lines. Methodology/Principal Findings: In a survey of different cell lines for efficient expression of platelet-derived growth factor-B (PDGF-B), beta-galactosidase (beta-gal) and green fluorescent protein (GFP) from plasmid vectors, PC3 was found to express at 5-50-fold higher levels compared to the bone metastatic prostate carcinoma cell line PC3BM and many other cell lines. Further, the efficiency of transfection and level of expression of the reporters in PC3 were comparable to that in 293T. Comparative analyses revealed that the high level expression of the reporters in the two cell lines was due to increased translational efficiency. While phosphatidic acid (PA)-mediated activation of mTOR, as revealed by drastic reduction in reporter expression by n-butanol, primarily contributed to the high level expression in PC3, multiple pathways involving PA, PI3K/Akt and ERK1/2 appear to contribute to the abundant reporter expression in 293T. Thus the extent of translational upregulation attained through the concerted activation of mTOR by multiple pathways in 293T could be achieved through its activation primarily by the PA pathway in PC3. Conclusions/Significance: Our studies reveal that the high-level expression of proteins from plasmid vectors is effected by translational up-regulation through mTOR activation via different signaling pathways in the two cell lines and that PC3 is as efficient as 293T for recombinant protein expression. Further, PC3 offers an advantage in that the level of expression of the protein can be regulated by simple addition of n-butanol to the culture medium.

The role of reciprocal regulation between TAL1 and miRNA expression in T-cell leukemia progression

Tese de doutoramento, Ciências Biomédicas (Biologia Celular e Molecular), Universidade de Lisboa, Faculdade de Medicina, 2015

Water deficits promote flowering in Rhododendron via regulation of pre and post initiation development

Flowering is generally considered to be advanced by water deficits in many woody perennial species. A long-standing paradigm being that as a plant senses severe environmental conditions resources are diverted away from vegetative growth and towards reproduction before death. It is demonstrated that in Rhododendron flowering is promoted under water deficit treatments. However, the promotion of flowering is not achieved via all increase in floral initiation, but through separate developmental responses. If regulated deficit irrigation (RDI) is imposed prior to the time of initiation, fewer vegetative nodes are formed before the apical meristems switch to floral initiation, and chronologically, floral initiation occurs earlier. Both RDI and partial rootzone drying (PRD) treatments stimulate the development of more flowers Oil each inflorescence if the treatments are continued after the plant has undergone floral initiation. However, floral initiation is inhibited by soil water deficits. If the soil water deficit continues beyond the stages of floral development then anthesis call occur prematurely oil the fully formed floral buds without a need for a winter chilling treatment. It is hypothesised that inhibition of floral initiation in plants experiencing severe soil water deficits results from the inhibitory action Of ABA transportation to the apical meristem from stressed roots. It is demonstrated that ABA applications to well-watered Rhododendron inhibit floral initiation. (c) 2008 Elsevier B.V. All rights reserved.

Impaired muscle Ca2+ and K+ regulation contribute to poor exercise performance post-lung transplantation

Lung transplant recipients (LTx) exhibit marked peripheral limitations to exercise. We investigated whether skeletal muscle Ca2+ and K+ regulation might be abnormal in eight LTx and eight healthy controls. Peak oxygen consumption and arterialized venous plasma [K+] (where brackets denote concentration) were measured during incremental exercise. Vastus lateralis muscle was biopsied at rest and analyzed for sarcoplasmic reticulum Ca2+ release, Ca2+ uptake, and Ca2+-ATPase activity rates; fiber composition; Na+-K+-ATPase (K+-stimulated 3-O-methylfluorescein phosphatase) activity and content ([3H]ouabain binding sites); as well as for [H+] and H+-buffering capacity. Peak oxygen consumption was 47% less in LTx (P < 0.05). LTx had lower Ca2+ release (34%), Ca2+ uptake (31%), and Ca2+-ATPase activity (25%) than controls (P < 0.05), despite their higher type II fiber proportion (LTx, 75.0 ± 5.8%; controls, 43.5 ± 2.1%). Muscle [H+] was elevated in LTx (P < 0.01), but buffering capacity was similar to controls. Muscle 3-O-methylfluorescein phosphatase activity was 31% higher in LTx (P < 0.05), but [3H]ouabain binding content did not differ significantly. However, during exercise, the rise in plasma [K+]-to-work ratio was 2.6-fold greater in LTx (P < 0.05), indicating impaired K+ regulation. Thus grossly subnormal muscle calcium regulation, with impaired potassium regulation, may contribute to poor muscular performance in LTx.

Regulation of sorting and post-Golgi trafficking of rhodopsin by its C-terminal sequence QVS(A)PA

Several mutations that cause severe forms of the human disease autosomal dominant retinitis pigmentosa cluster in the C-terminal region of rhodopsin. Recent studies have implicated the C-terminal domain of rhodopsin in its trafficking on specialized post-Golgi membranes to the rod outer segment of the photoreceptor cell. Here we used synthetic peptides as competitive inhibitors of rhodopsin trafficking in the frog retinal cell-free system to delineate the potential regulatory sequence within the C terminus of rhodopsin and model the effects of severe retinitis pigmentosa alleles on rhodopsin sorting. The rhodopsin C-terminal sequence QVS(A)PA is highly conserved among different species. Peptides that correspond to the C terminus of bovine (amino acids 324–348) and frog (amino acids 330–354) rhodopsin inhibited post-Golgi trafficking by 50% and 60%, respectively, and arrested newly synthesized rhodopsin in the trans-Golgi network. Peptides corresponding to the cytoplasmic loops of rhodopsin and other control peptides had no effect. When three naturally occurring mutations: Q344ter (lacking the last five amino acids QVAPA), V345M, and P347S were introduced into the frog C-terminal peptide, the inhibitory activity of the peptides was no longer detectable. These observations suggest that the amino acids QVS(A)PA comprise a signal that is recognized by specific factors in the trans-Golgi network. A lack of recognition of this sequence, because of mutations in the last five amino acids causing autosomal dominant retinitis pigmentosa, most likely results in abnormal post-Golgi membrane formation and in an aberrant subcellular localization of rhodopsin.

Calcium regulation of a slow post-spike hyperpolarization in vagal afferent neurons

Activation of distinct classes of potassium channels can dramatically affect the frequency and the pattern of neuronal firing. In a subpopulation of vagal afferent neurons (nodose ganglion neurons), the pattern of impulse activity is effectively modulated by a Ca2+-dependent K+ current. This current produces a post-spike hyperpolarization (AHPslow) that plays a critical role in the regulation of membrane excitability and is responsible for spike-frequency accommodation in these neurons. Inhibition of the AHPslow by a number of endogenous autacoids (e.g., histamine, serotonin, prostanoids, and bradykinin) results in an increase in the firing frequency of vagal afferent neurons from <0.1 to >10 Hz. After a single action potential, the AHPslow in nodose neurons displays a slow rise time to peak (0.3–0.5 s) and a long duration (3–15 s). The slow kinetics of the AHPslow are due, in part, to Ca2+ discharge from an intracellular Ca2+-induced Ca2+ release (CICR) pool. Action potential-evoked Ca2+ influx via either L or N type Ca2+ channels triggers CICR. Surprisingly, although L type channels generate 60% of action potential-induced CICR, only Ca2+ influx through N type Ca2+ channels can trigger the CICR-dependent AHPslow. These observations suggest that a close physical proximity exists between endoplasmic reticulum ryanodine receptors and plasma membrane N type Ca2+ channels and AHPslow potassium channels. Such an anatomical relation might be particularly beneficial for modulation of spike-frequency adaptation in vagal afferent neurons.

cAMP- and rapamycin-sensitive regulation of the association of eukaryotic initiation factor 4E and the translational regulator PHAS-I in aortic smooth muscle cells.

Incubating rat aortic smooth muscle cells with either platelet-derived growth factor BB (PDGF) or insulin-like growth factor I (IGF-I) increased the phosphorylation of PHAS-I, an inhibitor of the mRNA cap binding protein, eukaryotic initiation factor (eIF) 4E. Phosphorylation of PHAS-I promoted dissociation of the PHAS-I-eIF-4E complex, an effect that could partly explain the stimulation of protein synthesis by the two growth factors. Increasing cAMP with forskolin decreased PHAS-I phosphorylation and markedly increased the amount of eIF-4E bound to PHAS-I, effects consistent with an action of cAMP to inhibit protein synthesis. Both PDGF and IGF-I activated p70S6K, but only PDGF increased mitogen-activated protein kinase activity. Forskolin decreased by 50% the effect of PDGF on increasing p70S6K, and forskolin abolished the effect of IGF-I on the kinase. The effects of PDGF and IGF-I on increasing PHAS-I phosphorylation, on dissociating the PHAS-I-eIF-4E complex, and on increasing p70S6K were abolished by rapamycin. The results indicate that IGF-I and PDGF increase PHAS-I phosphorylation in smooth muscle cells by the same rapamycin-sensitive pathway that leads to activation of p70S6K.

Laws instructions and forms, for the regulation of the post-office department.

Mode of access: Internet.

Glutaredoxin-1 up-regulation induces soluble vascular endothelial growth factor receptor 1, attenuating post-ischemia limb revascularization

Glutaredoxin-1 (Glrx) is a cytosolic enzyme that regulates diverse cellular function by removal of GSH adducts from S-glutathionylated proteins including signaling molecules and transcription factors. Glrx is up-regulated during inflammation and diabetes. Glrx overexpression inhibits VEGF-induced endothelial cell (EC) migration. The aim was to investigate the role of up-regulated Glrx in EC angiogenic capacities and in vivo revascularization in the setting of hind limb ischemia. Glrx overexpressing EC from Glrx transgenic mice (TG) showed impaired migration and network formation and secreted higher level of soluble VEGF receptor 1 (sFlt), an antagonizing factor to VEGF. After hind limb ischemia surgery Glrx TG mice demonstrated impaired blood flow recovery, associated with lower capillary density and poorer limb motor function compared to wild type littermates. There were also higher levels of anti-angiogenic sFlt expression in the muscle and plasma of Glrx TG mice after surgery. Non-canonical Wnt5a is known to induce sFlt. Wnt5a was highly expressed in ischemic muscles and EC from Glrx TG mice, and exogenous Wnt5a induced sFlt expression and inhibited network formation in human microvascular EC. Adenoviral Glrx-induced sFlt in EC was inhibited by a competitive Wnt5a inhibitor. Furthermore, Glrx overexpression removed GSH adducts on p65 in ischemic muscle and EC, and enhanced nuclear factor kappa B (NF-kB) activity which was responsible for Wnt5a-sFlt induction. Taken together, up-regulated Glrx induces sFlt in EC via NF-kB -dependent Wnt5a, resulting in attenuated revascularization in hind limb ischemia. The Glrx-induced sFlt may be a part of mechanism of redox regulated VEGF signaling.

On the peculiar relevance of a fundamental dilemma of minimum-wage regulation in post-socialism – apropos of an international investigation

To the extent minimum-wage regulation is effective in fighting against excessive earnings handicaps of those at the lower end-tail of earnings distribution, it may have the side-effect of worsening their employment prospects. A demand-and-supply interpretation of data on the relative employment rate and earnings position of the least educated in the EU27 suggests that the resulting dilemma might be particularly relevant for minimum-wage policies in post-socialist countries.