Structural basis for translation termination by archaeal RF1 and GTP-bound EF1�� complex

When a stop codon appears at the ribosomal A site, the class I and II release factors (RFs) terminate translation. In eukaryotes and archaea, the class I and II RFs form a heterodimeric complex, and complete the overall translation termination process in a GTP-dependent manner. However, the structural mechanism of the translation termination by the class I and II RF complex remains unresolved. In archaea, archaeal elongation factor 1 alpha (aEF1α), a carrier GTPase for tRNA, acts as a class II RF by forming a heterodimeric complex with archaeal RF1 (aRF1). We report the crystal structure of the aRF1·aEF1α complex, the first active class I and II RF complex. This structure remarkably resembles the tRNA·EF–Tu complex, suggesting that aRF1 is efficiently delivered to the ribosomal A site, by mimicking tRNA. It provides insights into the mechanism that couples GTP hydrolysis by the class II RF to stop codon recognition and peptidyl-tRNA hydrolysis by the class I RF. We discuss the different mechanisms by which aEF1α recognizes aRF1 and aPelota, another aRF1-related protein and molecular evolution of the three functions of aEF1α.     

A G-quadruplex structure within the 5��-UTR of TRF2 mRNA represses translation in human cells

Telomeres protect chromosome ends from being recognized as double-stranded breaks. Telomeric function is ensured by the shelterin complex in which TRF2 protein is an essential player. The G-rich strand of telomere DNA can fold into G-quadruplex (G4) structure. Small molecules stabilizing G4 structures, named G4 ligands, have been shown to alter telomeric functions in human cells. In this study, we show that a guanine-rich RNA sequence located in the 5′-UTR region of the TRF2 mRNA (hereafter 91TRF2G) is capable of forming a stable quadruplex that causes a 2.8-fold decrease in the translation of a reporter gene in human cells, as compared to a mutant 5′-UTR unable to fold into G4. We also demonstrate that several highly selective G4 ligands, the pyridine dicarboxamide derivative 360A and bisquinolinium compounds Phen-DC(3) and Phen-DC(6), are able to bind the 91TRF2G:RNA sequence and to modulate TRF2 protein translation in vitro. Since the naturally occurring 5′-UTR TRF2:RNA G4 element was used here, which is conserved in several vertebrate orthologs, the present data substantiate a potential translational mechanism mediated by a G4 RNA motif for the downregulation of TRF2 expression.     

Splicing factor SRSF3 represses the translation of programmed cell death 4 mRNA by associating with the 5′-UTR region

Serine/arginine-rich splicing factor 3 (SRSF3), a member of the serine/arginine (SR)-rich family of proteins, regulates both alternative splicing of pre-mRNA and export of mature mRNA from the nucleus. Although its role in nuclear mRNA processing is well understood, the mechanism by which it alters the fate of cytoplasmic mRNA molecules remains elusive. Here, we provide evidence that SRSF3 not only regulates the alternative splicing pattern of programmed cell death 4 (PDCD4) mRNA, but also modulates its translational efficiency in the cytoplasm by lowering translation levels. We observed a marked increase in PDCD4 mRNA in translating polysome fractions upon silencing of SRSF3, and, conversely, ectopic overexpression of SRSF3 shifted PDCD4 mRNA into non-translating ribosomal fractions. In live cells, SRSF3 colocalized with PDCD4 mRNA in P-bodies (PBs), where translationally silenced mRNAs are deposited, and this localization was abrogated upon SRSF3 silencing. Furthermore, using two different reporter systems, we showed that SRSF3 interacts directly with PDCD4 mRNA and mediates translational repression by binding to the 5′-untranslated region (5′-UTR). In summary, our data suggest that the oncogenic potential of SRSF3 might be realized, in part, through the translational repression of PDCD4 mRNA.     

Polyamine modulon in yeast—Stimulation of COX4 synthesis by spermidine at the level of translation

We proposed that a group of genes whose expression is enhanced by polyamines at the level of translation in Escherichia coli and mammalian cells be referred to as a “polyamine modulon”. In Saccharomyces cerevisiae, proteins whose synthesis is enhanced by polyamines at the level of translation were searched for using a polyamine-requiring mutant of S. cerevisiae deficient in ornithine decarboxylase (YPH499 Δspe1). Addition of spermidine to the medium recovered the spermidine content and enhanced cell growth of the YPH499 Δspe1 mutant by 3–5-fold. Under these conditions, synthesis of COX4, one of the subunits of cytochrome C oxidase (complex IV), was enhanced by polyamines about 2.5-fold at the level of translation. Accordingly, the COX4 gene is the first member of a polyamine modulon in yeast. Polyamines enhanced COX4 synthesis through stimulation of the ribosome shunting of the stem–loop structures (hairpin structures) during the scanning of the 5′-untranslated region (5′-UTR) of COX4 mRNA by 40S ribosomal subunit-Met-tRNA_i complex.     

Activation of the EIF2α/ATF4 and ATF6 Pathways in DU-145 Cells by Boric Acid at the Concentration Reported in Men at the US Mean Boron Intake

We aimed to evaluate the association of exposure to lead with glycated hemoglobin (HbA1c), fasting glucose levels (FGLs), and the likelihood for dysglycemia. We accessed data from Canada Health and Measures Survey. General linear models were used to estimate the association between blood lead concentrations (BPb) and both HbA1c and FGLs, while controlling for confounders. Multivariate logistic regression was used for assessing the relation between BPb and the likelihood for dysglycemia. FGLs in participants with moderate BPb (2.5–5.0 μg/dL) were 1.03 (95 % CI 1.00–1.06) times higher compared with participants with BPb < 2.5 μg/dL. Equivalent figures for those with BPb ≥ 5.0 μg/dL were 1.10 (95 % CI 1.01–1.20) times, relative to the lowest stratum. This association was attenuated using HbA1c to define dysglycemia. Lead exposure was associated with the likelihood for neither FGLs ≥ 1.10 g/L nor HbA1c ≥ 5.7 %. The association between lead exposure and dysglycemia, if any, is likely to be very modest, at least at the population level.     

Differential Modulation of ��- and ��-Opioid Receptor Agonists by Endogenous RGS4 Protein in SH-SY5Y Cells

Regulator of G-protein signaling (RGS) proteins are a family of molecules that control the duration of G protein signaling. A variety of RGS proteins have been reported to modulate opioid receptor signaling. Here we show that RGS4 is abundantly expressed in human neuroblastoma SH-SY5Y cells that endogenously express μ- and δ-opioid receptors and test the hypothesis that the activity of opioids in these cells is modulated by RGS4. Endogenous RGS4 protein was reduced by ∼90% in SH-SY5Y cells stably expressing short hairpin RNA specifically targeted to RGS4. In these cells, the potency and maximal effect of δ-opioid receptor agonist (SNC80)-mediated inhibition of forskolin-stimulated cAMP accumulation was increased compared with control cells. This effect was reversed by transient transfection of a stable RGS4 mutant (HA-RGS4C2S). Furthermore, MAPK activation by SNC80 was increased in cells with knockdown of RGS4. In contrast, there was no change in the μ-opioid (morphine) response at adenylyl cyclase or MAPK. FLAG-tagged opioid receptors and HA-RGS4C2S were transiently expressed in HEK293T cells, and co-immunoprecipitation experiments showed that the δ-opioid receptor but not the μ-opioid receptor could be precipitated together with the stable RGS4. Using chimeras of the δ- and μ-opioid receptors, the C-tail and third intracellular domain of the δ-opioid receptor were suggested to be the sites of interaction with RGS4. The findings demonstrate a role for endogenous RGS4 protein in modulating δ-opioid receptor signaling in SH-SY5Y cells and provide evidence for a receptor-specific effect of RGS4.μ- and δ-opioid receptors are members of the G protein-coupled receptor family and interact with Gα_i/o proteins (1, 2). This results in signaling to a variety of downstream effectors, including adenylyl cyclase and the mitogen-activated protein kinase (MAPK)² cascade. Signaling of opioid receptors is regulated negatively by regulator of G protein signaling (RGS) proteins (3, 4). These are a family of molecules containing a “RGS consensus” domain that bind to Gα subunits and act as GTPase-accelerating proteins to increase the rate of GTP hydrolysis. This results in a decrease in the lifetime of the active Gα-GTP and free Gβγ subunits and limits signaling to downstream effectors (5–8). The mechanisms by which RGS proteins selectively modulate G protein-mediated receptor signal transduction pathways, especially opioid receptor signaling, are beginning to unfold (9–12). The foundation for the function and selectivity of RGS proteins in regulating opioid signaling lies in their ability to interact with opioid receptors and their cognate G proteins. In general, the selectivity or the preference of an RGS protein for a particular receptor is determined by a variety of factors, including tissue-specific expression and precise interaction with the intracellular domains of receptor proteins, G protein subunits, and effectors as well as other pathway-specific components (13).The effects of RGS proteins on opioid receptor signaling have been examined in several systems. The findings are not always consistent, probably due to the different methodologies used. It has been shown that members of the RZ, R4, and R7 subfamilies (7) of RGS proteins play crucial roles not only in terminating acute opioid agonist action but also in opioid receptor desensitization, internalization, recycling, and degradation (3, 14), thereby affecting opioid tolerance and dependence (15–18). Much work has been performed with RGS4, because it is a smaller RGS protein with a structure consisting of the RGS consensus (box) sequence and a small N terminus (19, 20). It also has a wide distribution in the brain, especially in brain regions important for opioid actions, including the striatum, locus coeruleus, dorsal horn of the spinal cord, and cerebral cortex (21). In vitro RGS4 has been shown to reverse δ-opioid receptor agonist-induced inhibition of cAMP synthesis in membranes prepared from NG108-15 cells (6). Overexpression of RGS4 in HEK293 cells also attenuated morphine-, [d-Ala²,N-Me-Phe⁴,Gly-ol⁵]enkephalin (DAMGO)-, and [d-Pen²,d-Pen⁵]enkephalin (DPDPE)-induced inhibition of adenylyl cyclase (22, 23). Co-expression of RGS4 with GIRK1/GIRK2 channels in Xenopus oocytes reduced the basal K⁺ current and accelerated the deactivation of GIRK channels activated by κ-opioid receptor agonist {"type":"entrez-nucleotide","attrs":{"text":"U69593","term_id":"4205069","term_text":"U69593"}}U69593 (24). Although these previous studies have provided evidence that RGS4 can negatively regulate opioid receptor signaling, they do not confirm a functional role for endogenous RGS4 in endogenous, nontransfected systems.Human neuroblastoma SH-SY5Y cells endogenously express μ- and δ-opioid receptors and a variety of Gα_i/o proteins (25–27). Here we show that RGS4 is abundantly found at both the mRNA and protein levels in these cells. Consequently, we used SH-SY5Y cells to examine the hypothesis that RGS4 negatively modulates opioid receptor signaling under physiological conditions. The endogenously expressed RGS4 level in SH-SY5Y cells was reduced using lentiviral delivery of short hairpin RNA (shRNA) targeting the RGS4 gene. This resulted in changes in δ- but not μ-opioid receptor-mediated signaling to adenylyl cyclase and the MAPK pathway. These findings argue for a selective interaction of RGS4 with the δ-opioid receptor. To test this, we expressed FLAG-tagged μ- and δ-opioid receptors together with a construct for a stable, proteosome-resistant RGS4 protein in HEK293T cells. Co-immunoprecipitation indicated that the δ-opioid but not the μ-opioid receptor was closely associated with RGS4, providing further evidence for a selective interaction between RGS4 and δ-opioid receptor signaling.     

Thermodynamics of 7-Methylguanosine Cation Stacking with Tryptophan upon mRNA 5′ Cap Binding to Translation Factor eIF4E

Abstract

All eukaryotic nuclear transcribed mRNAs possess the cap structure, consisting of 7-methylguanosine linked by the 5′-5′ triphosphate bridge to the first nucleoside. The goal of the present study is to dissect the enthalpy and entropy changes of association of the mRNA 5′ cap with eIF4E into contributions originating from the interaction of 7-methylguanosine with tryptophan. The model results are discussed in the context of the thermodynamic parameters for the association of eIF4E with synthetic cap analogues.     

miR-155-5p modulates malignant behaviors of hepatocellular carcinoma by directly targeting CTHRC1 and indirectly regulating GSK-3β-involved Wnt/β-catenin signaling

Background

Hepatocellular carcinoma (HCC) remains one of the most lethal cancers. MicroRNA-155 (miR-155) and collagen triple helix repeat containing 1 (CTHRC1) were found to be involved in hepatocarcinogenesis, but their detailed functions in HCC are unclear. Here, we aimed to investigate the underlying role of miR-155-5p and CTHRC1 in HCC.

Methods

miR-155-5p and CTHRC1 expression levels were detected by qRT-PCR, IHC and WB in HCC patients and cell lines. Dual-luciferase assay, qRT-PCR and WB were used to validate the target interaction between miR-155-5p and CTHRC1. Biological behaviors, including apoptosis, cell cycle progression, and cell proliferation, invasion and migration, were measured by flow cytometry, CCK-8 assay and Transwell tests. A xenograft model was established to examine the effects of miR-155-5p and CTHRC1 on tumor formation. WB was finally utilized to identify the role of GSK-3β-involved Wnt/β-catenin signaling in HCC growth and metastasis.

Results

Our results showed that miR-155-5p and CTHRC1 were down-regulated and up-regulated, respectively, in HCC patients and cell lines. Dual-luciferase assay verified that CTHRC1 was the direct target of miR-155-5p. Moreover, elevated miR-155-5p expression promoted apoptosis but suppressed cell cycle progression and cell proliferation, invasion and migration in vitro and facilitated tumor formation in vivo; elevated CTHRC1 expression abolished these biological effects. Additionally, miR-155-5p overexpression increased metastasis- and anti-apoptosis-related protein expression and decreased pro-apoptosis-related protein expression, while forced CTHRC1 expression conserved the expression of these proteins.

Conclusion

Altogether, our data suggested that miR-155-5p modulated the malignant behaviors of HCC by targeting CTHRC1 and regulating GSK-3β-involved Wnt/β-catenin signaling; thereby, miR-155-5p and CTHRC1 might be promising therapeutic targets for HCC patients.

     

Translation efficiency mediated by the 5′ untranslated region greatly affects protein production in <Emphasis Type="Italic">Aspergillus oryzae</Emphasis>

We demonstrate that the 5 untranslated region (5UTR) plays an important role in determining translation efficiency in Aspergillus oryzae, using a model -glucuronidase (GUS) expression system. Alterations in the 5UTR resulted in an increase in GUS activity of up to eight-fold, without affecting mRNA levels. Moreover, using the most effective 5UTR construct, we could achieve remarkable intracellular overproduction of GUS protein; and the GUS level reached more than 50% of the total soluble protein. This is the first experimental evidence indicating the feasibility of improving recombinant protein yield by promoting translation initiation in filamentous fungi.     

Oxidative Stress Decreases Phosphatidylinositol 4,5-Bisphosphate Levels by Deactivating Phosphatidylinositol- 4-phosphate 5-Kinase �� in a Syk-dependent Manner

Phosphatidylinositol 4,5-bisphosphate (PIP₂) has many essential functions and its homeostasis is highly regulated. We previously found that hypertonic stress increases PIP₂ by selectively activating the β isoform of the type I phosphatidylinositol phosphate 5-kinase (PIP5Kβ) through Ser/Thr dephosphorylation and promoting its translocation to the plasma membrane. Here we report that hydrogen peroxide (H₂O₂) also induces PIP5Kβ Ser/Thr dephosphorylation, but it has the opposite effect on PIP₂ homeostasis, PIP5Kβ function, and the actin cytoskeleton. Brief H₂O₂ treatments decrease cellular PIP₂ in a PIP5Kβ-dependent manner. PIP5Kβ is tyrosine phosphorylated, dissociates from the plasma membrane, and has decreased lipid kinase activity. In contrast, the other two PIP5K isoforms are not inhibited by H₂O₂. We identified spleen tyrosine kinase (Syk), which is activated by oxidants, as a candidate PIP5Kβ kinase in this pathway, and mapped the oxidant-sensitive tyrosine phosphorylation site to residue 105. The PIP5KβY105E phosphomimetic is catalytically inactive and cytosolic, whereas the Y105F non-phosphorylatable mutant has higher intrinsic lipid kinase activity and is much more membrane associated than wild type PIP5Kβ. These results suggest that during oxidative stress, as modeled by H₂O₂ treatment, Syk-dependent tyrosine phosphorylation of PIP5Kβ is the dominant post-translational modification that is responsible for the decrease in cellular PIP₂.Oxygen-derived free radicals are by-products of metabolic reactions in eukaryotic cells. Reactive oxygen species (ROS)⁴ act as endogenous signaling molecules (1). However, excessive ROS production leads to deleterious effects on cellular homeostasis by inducing DNA damage, lipid/protein oxidation, and ultimately apoptosis or necrosis. Acute and chronic oxidative stress have been implicated in the pathophysiology of shock and sepsis associated with traumatic injuries such as massive thermal burn (2–4), Alzheimer disease, diabetes mellitus, and atherosclerosis (5–7).Phosphatidylinositol 4,5-bisphosphate (PIP₂) has emerged as an integral component of the stress response. This is concordant with its essential role in the regulation of the actin cytoskeleton, endocytosis, exocytosis, plasma membrane (PM) scaffolding, and ion channels/transporter (8). PIP₂ is also essential for InsP₃-mediated Ca²⁺ generation, protein kinase C activation, and PIP₃ generation (9, 10). PIP₂ synthesis is depressed in the heart sarcolemma during oxidative stress, suggesting that PIP₂ depletion may contribute to cardiac dysfunctions (11). Recently, Divecha and colleagues (12) reported that prolonged (many hours) treatment of HeLa cells with hydrogen peroxide (H₂O₂) induces apoptosis by depleting PIP₂. Apoptosis can be attenuated by overexpression of a type I phosphatidylinositol-4-phosphate 5-kinase (PIP5Kβ). We found using isoform-specific PIP5K knockdown by RNA interference (RNAi) that PIP5Kβ synthesizes a large fraction of the ambient PIP₂ pool in HeLa cells (13). Hypertonicity is another type of stress that increases PIP₂ and may be protective against cell injury (14, 15) by activating PIP5Kβ through Ser/Thr dephosphorylation (16). This effect is specific for PIP5Kβ, because depletion of the other two PIP5K isoforms (α and γ) individually does not substantially abrogate the hypertonicity induced PIP₂ increase.In the present study, we used H₂O₂ to model oxidative stress in tissue culture cells, and examined the effect on PIP₂ homeostasis and PIP5Kβ function. We found that a brief H₂O₂ treatment decreases cellular PIP₂ and inactivates PIP5Kβ through tyrosine phosphorylation. We identified spleen tyrosine kinase (Syk) as a candidate kinase in this pathway. Syk is a member of the Syk/Zap-70 nonreceptor tyrosine kinase family that is abundant in hematopoietic cells (17) but is also found in nonhematopoietic lineages (18), including HeLa and COS cells (19, 20).     

TRAIL death receptors DR4 and DR5 mediate cerebral microvascular endothelial cell apoptosis induced by oligomeric Alzheimer's A��

Vascular deposition of amyloid-β (Aβ) in sporadic and familial Alzheimer''s disease, through poorly understood molecular mechanisms, leads to focal ischemia, alterations in cerebral blood flow, and cerebral micro-/macro-hemorrhages, significantly contributing to cognitive impairment. Here, we show that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) death receptors DR4 and DR5 specifically mediate oligomeric Aβ induction of extrinsic apoptotic pathways in human microvascular cerebral endothelial cells with activation of both caspase-8 and caspase-9. The caspase-8 inhibitor cellular FLICE-like inhibitory protein (cFLIP) is downregulated, and mitochondrial paths are engaged through BH3-interacting domain death agonist (Bid) cleavage. Upregulation of DR4 and DR5 and colocalization with Aβ at the cell membrane suggests their involvement as initiators of the apoptotic machinery. Direct binding assays using receptor chimeras confirm the specific interaction of oligomeric Aβ with DR4 and DR5 whereas apoptosis protection achieved through RNA silencing of both receptors highlights their active role in downstream apoptotic pathways unveiling new targets for therapeutic intervention.