scholarly journals The suppressive cap-binding-complex factor 4EIP is required for normal differentiation

2018 ◽  
Author(s):  
Monica Terrao ◽  
Kevin Kamanyi Marucha ◽  
Elisha Mugo ◽  
Dorothea Droll ◽  
Ihor Minia ◽  
...  
Keyword(s):  
Growth Defect ◽  
Binding Motif ◽  
Mrna Levels ◽  
Suppressive Activity ◽  
Mrna Content ◽  
Cap Binding Complex ◽  
Normal Differentiation ◽  
Mrna Binding ◽  
Stumpy Form

Summary/AbstractTrypanosoma brucei live in mammals as bloodstream forms and in the Tsetse midgut as procyclic forms. Differentiation from one form to the other proceeds via a growth-arrested stumpy form with low mRNA content and translation. The parasites have six eIF4Es and five eIF4Gs. EIF4E1 pairs with the mRNA-binding protein 4EIP but not with any EIF4G. EIF4E1 and 4EIP each inhibit expression when tethered to a reporter mRNA. The 4E-binding motif in 4EIP is required for the interaction with EIF4E1 both in vivo and in a 2-hybrid assay, but not for the suppressive activity of 4EIP when tethered. However, the suppressive activity of EIF4E1 when tethered requires 4EIP. Correspondingly, in growing bloodstream forms, 4EIP is preferentially associated with unstable mRNAs. Trypanosomes lacking 4EIP have a marginal growth disadvantage as cultured bloodstream or procyclic forms. Bloodstream forms without 4EIP cannot make differentiation-competent stumpy forms, but the defect can be complemented by a truncated 4EIP that does not interact with EIF4E1. Bloodstream forms lacking EIF4E1 have a growth defect but can differentiate. We suggest that 4EIP and EIF4E1 fine-tune mRNA levels in growing cells, and that 4EIP is required for mRNA suppression during differentiation to the stumpy form.

Two Novel Trans-Acting Factors Dictate the High Cytoplasmic Stability of β-Globin mRNA

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 644-644 ◽  
Author(s):  
Sebastiaan van Zalen ◽  
J. Eric Russell
Keyword(s):  
Binding Site ◽  
Globin Gene ◽  
K562 Cells ◽  
Binding Motif ◽  
Target Sequence ◽  
Globin Mrna ◽  
Erythroid Progenitor ◽  
Intact Cells ◽  
Mrna Binding

Abstract Abstract 644 The efficient accumulation of hemoglobin in mature erythrocytes is critically dependent upon the high stabilities of mRNAs encoding human α- and β-globin proteins. These mRNAs are likely to be stabilized by interactions between one or more trans-acting regulatory factors that target defined cis-acting elements within their 3′UTRs. Several ubiquitous factors that are known to bind to the β-globin 3′UTR (including αCP, PTBP1, and nucleolin) are largely restricted to the nucleus and therefore unlikely to contribute to regulatory processes affecting β-globin mRNA in the cytoplasm. Consequently, we conducted a series of experiments that identify and characterize mRNA-binding factors that dictate the properties of β-globin mRNA in the cytoplasm of erythroid progenitor cells. Using electrophoretic gel mobility shift analyses (EMSA), we defined a characteristic mRNP complex that assembles on the β-globin 3′UTR in cytoplasmic extract–but not nuclear extract–prepared from erythroid K562 cells. This mRNP ‘β-complex’ appears to be erythroid-specific, as it fails to assemble in extracts prepared from non-erythroid HeLa or HEK cells. The 3′UTR binding site for the β-complex was identified using an EMSA-competition approach; remarkably, the target sequence is encompassed within a 12-nt region previously identified as a functional determinant of β-globin mRNA stability in in vivo analyses. Additional experiments fine-mapped the β-complex binding site to a GGGGG pentanucleotide motif within the mRNA-stabilizing region. The functional importance of the pentanucleotide was illustrated by mRNA decay experiments in intact erythroid K562 cells showing that full-length β-globin mRNAs are destabilized by introduction of the same GGGGG->CCGGG mutation that ablates β-complex assembly in EMSA analyses. To identify trans-factors that comprise the β-complex, we performed affinity chromatography using ssDNA probes corresponding to the β-complex binding motif. The native 3′UTR probe retained 42- and 47-kDa proteins, while a probe carrying the CCGGG mutation failed to bind either factor. Subsequent LC/MS/MS analyses identified the two proteins as YB-1 and AUF-1. The identities of these two mRNA-binding factors, which have previously been implicated in the post-transcriptional regulation of heterologous mRNAs, were subsequently confirmed by immunoblot of the protein-DNA complexes. Subsequent analyses suggested a functional role for both factors: EMSA supershift experiments confirmed that YB-1 is a component of the β-complex, and RNA immunoprecipitation analyses demonstrated that both YB-1 and AUF-1 specifically bind to β-globin mRNA in vivo in intact erythroid K562 cells. Collectively, these data identify two novel trans-acting factors that bind to cytoplasmic β-globin mRNA in an erythroid-specific fashion, at a site that dictates its stability in intact cells. We are currently engaged in siRNA knock-down experiments to validate experiments that suggest the importance of these trans-acting factors to the constitutive cytoplasmic stability of β-globin mRNA, as well as structural analyses intended to define RNA-protein and protein-protein interactions that are critical to normal functioning of the β-complex. The results of these experiments have obvious implications for the design of novel therapies for patients with congenital disorders of β-globin gene expression, including sickle cell disease and β thalassemia. Disclosures: No relevant conflicts of interest to declare.

Expression of kinin B1 receptor in fresh or cultured rabbit aortic smooth muscle: role of NF-κB

2002 ◽  
Vol 283 (1) ◽  
pp. H227-H237 ◽  
Author(s):  
Thierry Sabourin ◽  
Guillaume Morissette ◽  
Johanne Bouthillier ◽  
Luc Levesque ◽  
François Marceau
Keyword(s):  
Smooth Muscle ◽  
Rabbit Aorta ◽  
Aortic Tissue ◽  
Mrna Levels ◽  
Kinin B1 Receptor ◽  
Epidermal Growth ◽  
Fetal Bovine ◽  
Cultured Smooth Muscle Cells ◽  
Mrna Binding

Kinin B1receptor (B1R) expression and the importance of the transcription factor nuclear factor (NF)-κB in this process were evaluated in models based on the rabbit aorta: freshly isolated tissue (postisolation induction) and cultured smooth muscle cells (SMCs). A 3-h incubation of freshly isolated tissues determined a sharp B1R mRNA increase (RT-PCR). Coincubation of tissues with a stimulus (interleukin-1β, fetal bovine serum, epidermal growth factor, or cycloheximide) further increased mRNA levels. Cultured SMCs possessed a basal population of surface B1Rs ([3H]Lys-des-Arg9-bradykinin binding) that was upregulated by treatments with the same set of stimuli (binding, mRNA, nuclear runon). Pharmacological inhibitors of NF-κB (MG-132, BAY 11-7082, dexamethasone) or actinomycin D reduced the postisolation induction of B1Rs in fresh aortic tissue (contractility or mRNA) and the cytokine effect on cells (mRNA, binding). NF-κB may be a common mediator of various stimuli that increase B1R gene transcription in the rabbit aorta, including tissue isolation, but cycloheximide also stabilizes B1R mRNA. The SMC models faithfully mimic the in vivo situation with regard to B1R regulation.

scholarly journals Structure-Function Analysis of Yeast mRNA Cap Methyltransferase and High-Copy Suppression of Conditional Mutants by AdoMet Synthase and the Ubiquitin Conjugating Enzyme Cdc34p

Genetics ◽  
2000 ◽  
Vol 155 (4) ◽  
pp. 1561-1576
Author(s):  
Beate Schwer ◽  
Nayanendu Saha ◽  
Xiangdong Mao ◽  
Hsiao-Wang Chen ◽  
Stewart Shuman
Keyword(s):  
Function Analysis ◽  
Growth Defect ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Mrna Levels ◽  
Ubiquitin Conjugating Enzyme ◽  
Ubiquitin Conjugation ◽  
Yeast Genes ◽  
Conjugating Enzyme

Abstract Here we present a genetic analysis of the yeast cap-methylating enzyme Abd1p. To identify individual amino acids required for Abd1p function, we introduced alanine mutations at 35 positions of the 436-amino acid yeast protein. Two new recessive lethal mutations, F256A and Y330A, were identified. Alleles F256L and Y256L were viable, suggesting that hydrophobic residues at these positions sufficed for Abd1p function. Conservative mutations of Asp-178 established that an acidic moiety is essential at this position (i.e., D178E was viable whereas D178N was not). Phe-256, Tyr-330, and Asp-178 are conserved in all known cellular cap methyltransferases. We isolated temperature-sensitive abd1 alleles and found that abd1-ts cells display a rapid shut-off of protein synthesis upon shift to the restrictive temperature, without wholesale reduction in steady-state mRNA levels. These in vivo results are consistent with classical biochemical studies showing a requirement for the cap methyl group in cap-dependent translation. We explored the issue of how cap methylation might be regulated in vivo by conducting a genetic screen for high-copy suppressors of the ts growth defect of abd1 mutants. The identification of the yeast genes SAM2 and SAM1, which encode AdoMet synthase, as abd1 suppressors suggests that Abd1p function can be modulated by changes in the concentration of its substrate AdoMet. We also identified the ubiquitin conjugating enzyme Cdc34p as a high-copy abd1 suppressor. We show that mutations of Cdc34p that affect its ubiquitin conjugation activity or its capacity to interact with the E3-SCF complex abrogate its abd1 suppressor function. Moreover, the growth defect of abd1 mutants is exacerbated by cdc34-2. These findings suggest a novel role for Cdc34p in gene expression and engender a model whereby cap methylation or cap utilization is negatively regulated by a factor that is degraded when Cdc34p is overexpressed.

Abstract 9772: Antagonism of Rosuvastatin-induced Elevation of Circulating PCSK9 in Hamsters by Liver-specific Knockdown of HNF1α or HNF1β

Circulation ◽  
2015 ◽  
Vol 132 (suppl_3) ◽  
Author(s):  
Bin Dong ◽  
Amar B Singh ◽  
Vikram R Shende ◽  
Jingwen Liu
Keyword(s):  
Ldl Cholesterol ◽  
Hdl Cholesterol ◽  
Proximal Promoter ◽  
In Vivo Studies ◽  
Expression Vectors ◽  
Binding Motif ◽  
Mrna Levels ◽  
Shrna Expression ◽  
The Impact

Proprotein convertase subtilisin/kexin type 9 (PCSK9) induces hepatic LDL receptor (LDLR) degradation and lowers the removal of LDL-cholesterol from circulation. Thus, PCSK9 has become an important therapeutic target for the treatment of hypercholesterolemia. The transcription of PCSK9 is prominently controlled by the HNF1 binding motif embedded in its proximal promoter region that binds to HNF1α or HNF1β as homodimers or heterodimers. Our previous in vivo studies showed that only HNF1α but not HNF1β regulates PCSK9 transcription in mouse liver. Statin has been shown to elevate circulating PCSK9 levels through stimulating PCSK9 gene transcription, which reduces the clinical efficacy of statin in LDL-cholesterol reduction. In this study, we utilized adenoviral shRNA expression vectors (Ad-shHNF1α and Ad-shHNF1β) to generate liver specific knockdown of HNF1α or HNF1β in hamsters to examine the impact of reduced expression of HNF1 transcription factors on statin-induced elevation of PCSK9. Administration of rosuvastatin (RSV) at a daily dose of 15 mg/kg to hamsters infected with a control adenovirus significantly increased liver PCSK9 mRNA levels by 94% and serum PCSK9 levels by 37%. However, injection of Ad-shHNF1α into hamsters largely abolished the RSV-induced elevation of PCSK9 serum levels and hepatic mRNA levels, which was accompanied by a significant increase in liver LDLR protein abundance by 64% as compared to hamsters injected with control virus. Surprisingly, injection of Ad-shHNF1β produced similar inhibitory effects on the RSV-induced PCSK9 expression as that of Ad-shHNF1α. This was different from the negative results of HNF1β knockdown conducted in mice. In addition to changes in PCSK9 levels, we observed a modest but significant reduction in serum non-HDL cholesterol level after knockdown of HNF1α (27%) or HNF1β (32%) in hamsters treated with RSV. Altogether, our study demonstrates that unlike mice, both HNF1α and HNF1β are positive regulators of hepatic PCSK9 transcription in hamster species and that transient, liver specific knockdown of either HNF1α or HNF1β could antagonize the RSV-induced elevation of serum PCSK9 and non-HDL cholesterol levels.

Clearance of Desialylated Platelets by the Hepatic Asialoglycoprotein Receptor Regulates TPO Homeostasis in Vivo.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2170-2170
Author(s):  
Renata Grozovsky ◽  
Antonija Jurak Begonja ◽  
John H. Hartwig ◽  
Karin M. Hoffmeister
Keyword(s):  
Liver Tissue ◽  
Conflicts Of Interest ◽  
Asialoglycoprotein Receptor ◽  
Published Data ◽  
Mrna Levels ◽  
Mrna Synthesis ◽  
Platelet Destruction ◽  
Mrna Content ◽  
Functional Receptor

Abstract Abstract 2170 The human body produces and removes 1011 platelets daily to maintain a normal steady-state platelet count, and the level of production can be greatly increased under conditions of platelet destruction. Here, we provide the experimental evidence that platelets with impaired Siaa2–3Galb1–4GlcNAc (LacNAc) structures are removed by the hepatic asialoglycoprotein receptor Asgr1/2, indicating that survival of platelets is intimately tied to surface glycans. Mice lacking Asgr2 subunit that is necessary to assemble a functional receptor have increased platelet survival (t1/2 = 49.5 ± 2h) compared to wild type mice (t1/2 = 31 ± 4h). Surprisingly, platelets from Asgr2-null mice have diminished surface sialic acid, as evidenced by lectins that bind exposed Gal moieties. Hence, desialylated platelets circulate in Asgr2-null mice. Besides “terminating” platelet circulation, the liver is the main source of thrombopoietin (TPO), the major hormone regulating platelet production. We hypothesized that desialylated platelet uptake by hepatic Asgr1/2 would affects TPO mRNA synthesis and have found that liver tissue from Asgr2-null mice has a 40% decrease in TPO mRNA levels compared to liver tissue from WT mice. In contrast, ST3Gal4-null mice, which have high rates of platelet turnover and increased desialylated platelet uptake by the Asgr2, have a 30% increase in TPO mRNA content in their livers. Both plasma TPO levels and platelet TPO contents are similarly altered in both mutant mice. In contrast, and in agreement with published data, antibody-mediated platelet clearance did not affect hepatic TPO mRNA levels. Taken together, these data show that the clearance of desialylated platelets by the hepatic Asgr1/2 regulates TPO homeostasis in vivo. Disclosures: No relevant conflicts of interest to declare.

SAC3B is a target of CML19, the centrin 2 of Arabidopsis thaliana

2020 ◽  
Vol 477 (1) ◽  
pp. 173-189 ◽  
Author(s):  
Marco Pedretti ◽  
Carolina Conter ◽  
Paola Dominici ◽  
Alessandra Astegno
Keyword(s):  
Excision Repair ◽  
Complex Structure ◽  
Binding Motif ◽  
C Terminus ◽  
Repair Protein ◽  
Nucleotide Excision ◽  
Ef Hand ◽  
Molecular Determinants ◽  
Structure In Solution

Arabidopsis centrin 2, also known as calmodulin-like protein 19 (CML19), is a member of the EF-hand superfamily of calcium (Ca2+)-binding proteins. In addition to the notion that CML19 interacts with the nucleotide excision repair protein RAD4, CML19 was suggested to be a component of the transcription export complex 2 (TREX-2) by interacting with SAC3B. However, the molecular determinants of this interaction have remained largely unknown. Herein, we identified a CML19-binding site within the C-terminus of SAC3B and characterized the binding properties of the corresponding 26-residue peptide (SAC3Bp), which exhibits the hydrophobic triad centrin-binding motif in a reversed orientation (I8W4W1). Using a combination of spectroscopic and calorimetric experiments, we shed light on the SAC3Bp–CML19 complex structure in solution. We demonstrated that the peptide interacts not only with Ca2+-saturated CML19, but also with apo-CML19 to form a protein–peptide complex with a 1 : 1 stoichiometry. Both interactions involve hydrophobic and electrostatic contributions and include the burial of Trp residues of SAC3Bp. However, the peptide likely assumes different conformations upon binding to apo-CML19 or Ca2+-CML19. Importantly, the peptide dramatically increases the affinity for Ca2+ of CML19, especially of the C-lobe, suggesting that in vivo the protein would be Ca2+-saturated and bound to SAC3B even at resting Ca2+-levels. Our results, providing direct evidence that Arabidopsis SAC3B is a CML19 target and proposing that CML19 can bind to SAC3B through its C-lobe independent of a Ca2+ stimulus, support a functional role for these proteins in TREX-2 complex and mRNA export.

Nanocurcumin: A Double-Edged Sword for Microcancers

2020 ◽  
Vol 26 (45) ◽  
pp. 5783-5792
Author(s):  
Kholood Abid Janjua ◽  
Adeeb Shehzad ◽  
Raheem Shahzad ◽  
Salman Ul Islam ◽  
Mazhar Ul Islam
Keyword(s):  
Intracellular Signaling ◽  
Dosage Forms ◽  
The Body ◽  
In Vivo Studies ◽  
Mrna Levels ◽  
Anticancer Activities ◽  
Road Map ◽  
Anticancer Effects

There is compelling evidence that drug molecules isolated from natural sources are hindered by low systemic bioavailability, poor absorption, and rapid elimination from the human body. Novel approaches are urgently needed that could enhance the retention time as well as the efficacy of natural products in the body. Among the various adopted approaches to meet this ever-increasing demand, nanoformulations show the most fascinating way of improving the bioavailability of dietary phytochemicals through modifying their pharmacokinetics and pharmacodynamics. Curcumin, a yellowish pigment isolated from dried ground rhizomes of turmeric, exhibits tremendous pharmacological effects, including anticancer activities. Several in vitro and in vivo studies have shown that curcumin mediates anticancer effects through the modulation (upregulation and/or downregulations) of several intracellular signaling pathways both at protein and mRNA levels. Scientists have introduced multiple modern techniques and novel dosage forms for enhancing the delivery, bioavailability, and efficacy of curcumin in the treatment of various malignancies. These novel dosage forms include nanoparticles, liposomes, micelles, phospholipids, and curcumin-encapsulated polymer nanoparticles. Nanocurcumin has shown improved anticancer effects compared to conventional curcumin formulations. This review discusses the underlying molecular mechanism of various nanoformulations of curcumin for the treatment of different cancers. We hope that this study will make a road map for preclinical and clinical investigations of cancer and recommend nano curcumin as a drug of choice for cancer therapy.

scholarly journals TRIM27 interacts with Iκbα to promote the growth of human renal cancer cells through regulating the NF-κB pathway

BMC Cancer ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Chengwu Xiao ◽  
Wei Zhang ◽  
Meimian Hua ◽  
Huan Chen ◽  
Bin Yang ◽  
...  
Keyword(s):  
Cell Lines ◽  
Prognostic Indicator ◽  
The Cancer Genome Atlas ◽  
Mrna Levels ◽  
Loss Of Function ◽  
Protein Levels ◽  
Kaplan Meier ◽  
Cancer Genome Atlas

Abstract Background The tripartite motif (TRIM) family proteins exhibit oncogenic roles in various cancers. The roles of TRIM27, a member of the TRIM super family, in renal cell carcinoma (RCC) remained unexplored. In the current study, we aimed to investigate the clinical impact and roles of TRIM27 in the development of RCC. Methods The mRNA levels of TRIM27 and Kaplan–Meier survival of RCC were analyzed from The Cancer Genome Atlas database. Real-time PCR and Western blotting were used to measure the mRNA and protein levels of TRIM27 both in vivo and in vitro. siRNA and TRIM27 were exogenously overexpressed in RCC cell lines to manipulate TRIM27 expression. Results We discovered that TRIM27 was elevated in RCC patients, and the expression of TRIM27 was closely correlated with poor prognosis. The loss of function and gain of function results illustrated that TRIM27 promotes cell proliferation and inhibits apoptosis in RCC cell lines. Furthermore, TRIM27 expression was positively associated with NF-κB expression in patients with RCC. Blocking the activity of NF-κB attenuated the TRIM27-mediated enhancement of proliferation and inhibition of apoptosis. TRIM27 directly interacted with Iκbα, an inhibitor of NF-κB, to promote its ubiquitination, and the inhibitory effects of TRIM27 on Iκbα led to NF-κB activation. Conclusions Our results suggest that TRIM27 exhibits an oncogenic role in RCC by regulating NF-κB signaling. TRIM27 serves as a specific prognostic indicator for RCC, and strategies targeting the suppression of TRIM27 function may shed light on future therapeutic approaches.

scholarly journals Glucolipotoxicity and GLP-1 secretion

2021 ◽  
Vol 9 (1) ◽  
pp. e001905
Author(s):  
Jung-Hee Hong ◽  
Dae-Hee Kim ◽  
Moon-Kyu Lee
Keyword(s):  
Glucose Transporter ◽  
Cell Function ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Gene Expressions ◽  
Simultaneous Treatment ◽  
L Cells ◽  
Triglyceride Accumulation

IntroductionThe concept of glucolipotoxicity refers to the combined, deleterious effects of elevated glucose and/or fatty acid levels.Research design and methodsTo investigate the effects of chronic glucolipotoxicity on glucagon-like peptide-1-(7-36) amide (GLP-1) secretion, we generated glucolipotoxic conditions in human NCI-H716 enteroendocrine cells using either 5 or 25 mM glucose with or without 500 µM palmitate for 72 hours. For in vivo study, we have established a chronic nutrient infusion model in the rat. Serial blood samples were collected for 2 hours after the consumption of a mixed meal to evaluate insulin sensitivity and β-cell function.ResultsChronic glucolipotoxic conditions decreased GLP-1 secretion and the expressions of pCREB, pGSK3β, β-catenin, and TCF7L2 in NCI-H716 cells. Glucolipotoxicity conditions reduced glucose transporter expression, glucose uptake, and nicotinamide-adenine dinucleotide phosphate (NADPH) levels in L-cells, and increased triglyceride accumulation. In contrast, PPARα and ATP levels were reduced, which correlated well with decreased levels of SUR1 and Kir6.2, cAMP contents and expressions of pCAMK2, EPAC and PKA. We also observed an increase in reactive oxygen species production, UCP2 expression and Complex I activity. Simultaneous treatment with insulin restored the GLP-1 secretion. Glucolipotoxic conditions decreased insulin secretion in a time-dependent manner in INS-1 cells, which was recovered with exendin-4 cotreatment. Glucose and SMOFlipid infusion for 6 hours decreased GLP-1 secretion and proglucagon mRNA levels as well as impaired the glucose tolerance, insulin and C-peptide secretion in rats.ConclusionThese results provide evidence for the first time that glucolipotoxicity could affect GLP-1 secretion through changes in glucose and lipid metabolism, gene expressions, and proglucagon biosynthesis and suggest the interrelationship between glucolipotoxicities of L-cells and β-cells which develops earlier than that of L-cells.

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