An A + U-rich element RNA-binding factor regulates c-myc mRNA stability in vitro

1991 ◽  
Vol 11 (5) ◽  
pp. 2460-2466 ◽  
Author(s):  
G Brewer
Keyword(s):  
Cellular Response ◽  
Mrna Decay ◽  
Rna Binding ◽  
Binding Capacity ◽  
Proteinase K ◽  
Rapid Turnover ◽  
A Cell ◽  
Degradation Activity ◽  
Proteinase K Treatment

Transient expression of some proto-oncogenes, cytokines, and transcription factors occurs as a cellular response to growth factors, 12-O-tetradecanoylphorbol-13-acetate, antigen stimulation, or inflammation. Expression of these genes is mediated in part by the rapid turnover of their mRNAs. A + U-rich elements in the 3' untranslated regions of these mRNAs serve as one recognition signal targeting the mRNAs for rapid degradation. I report the identification of a cytosolic factor that both binds to the proto-oncogene c-myc A + U-rich element and specifically destabilizes c-myc mRNA in a cell-free mRNA decay system which reconstitutes mRNA decay processes found in cells. Proteinase K treatment of the factor abolishes its c-myc mRNA degradation activity without affecting its RNA-binding capacity. Thus, RNA substrate binding and degradation appear to be separable functions. These findings should aid in understanding how the cell selectively targets mRNAs for rapid turnover.

scholarly journals An A + U-rich element RNA-binding factor regulates c-myc mRNA stability in vitro.

1991 ◽  
Vol 11 (5) ◽  
pp. 2460-2466 ◽  
Author(s):  
G Brewer
Keyword(s):  
Cellular Response ◽  
Mrna Decay ◽  
Rna Binding ◽  
Binding Capacity ◽  
Proteinase K ◽  
Rapid Turnover ◽  
A Cell ◽  
Degradation Activity ◽  
Proteinase K Treatment

Transient expression of some proto-oncogenes, cytokines, and transcription factors occurs as a cellular response to growth factors, 12-O-tetradecanoylphorbol-13-acetate, antigen stimulation, or inflammation. Expression of these genes is mediated in part by the rapid turnover of their mRNAs. A + U-rich elements in the 3' untranslated regions of these mRNAs serve as one recognition signal targeting the mRNAs for rapid degradation. I report the identification of a cytosolic factor that both binds to the proto-oncogene c-myc A + U-rich element and specifically destabilizes c-myc mRNA in a cell-free mRNA decay system which reconstitutes mRNA decay processes found in cells. Proteinase K treatment of the factor abolishes its c-myc mRNA degradation activity without affecting its RNA-binding capacity. Thus, RNA substrate binding and degradation appear to be separable functions. These findings should aid in understanding how the cell selectively targets mRNAs for rapid turnover.

scholarly journals PRMT7 regulates RNA-binding capacity and protein stability in Leishmania parasites

2020 ◽  
Vol 48 (10) ◽  
pp. 5511-5526
Author(s):  
Tiago R Ferreira ◽  
Adam A Dowle ◽  
Ewan Parry ◽  
Eliza V C Alves-Ferreira ◽  
Karen Hogg ◽  
...  
Keyword(s):  
Protein Modification ◽  
Transcriptional Control ◽  
Leishmania Major ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Capacity ◽  
Arginine Methylation ◽  
Mrna Metabolism

Abstract RNA binding proteins (RBPs) are the primary gene regulators in kinetoplastids as transcriptional control is nearly absent, making Leishmania an exceptional model for investigating methylation of non-histone substrates. Arginine methylation is an evolutionarily conserved protein modification catalyzed by Protein aRginine Methyl Transferases (PRMTs). The chromatin modifier PRMT7 is the only Type III PRMT found in higher eukaryotes and a restricted number of unicellular eukaryotes. In Leishmania major, PRMT7 is a cytoplasmic protein implicit in pathogenesis with unknown substrates. Using comparative methyl-SILAC proteomics for the first time in protozoa, we identified 40 putative targets, including 17 RBPs hypomethylated upon PRMT7 knockout. PRMT7 can modify Alba3 and RBP16 trans-regulators (mammalian RPP25 and YBX2 homologs, respectively) as direct substrates in vitro. The absence of PRMT7 levels in vivo selectively reduces Alba3 mRNA-binding capacity to specific target transcripts and can impact the relative stability of RBP16 in the cytoplasm. RNA immunoprecipitation analyses demonstrate PRMT7-dependent methylation promotes Alba3 association with select target transcripts and thus indirectly stabilizes mRNA of a known virulence factor, δ-amastin surface antigen. These results highlight a novel role for PRMT7-mediated arginine methylation of RBP substrates, suggesting a regulatory pathway controlling gene expression and virulence in Leishmania. This work introduces Leishmania PRMTs as epigenetic regulators of mRNA metabolism with mechanistic insight into the functional manipulation of RBPs by methylation.

Contribution of a 12 kDa protein to the angiotensin II-induced stabilization of angiotensinogen mRNA: interaction with the 3′ untranslated mRNA

1995 ◽  
Vol 14 (2) ◽  
pp. 209-226 ◽  
Author(s):  
C Klett ◽  
M Bader ◽  
M Schwemmle ◽  
D Ganten ◽  
E Hackenthal
Keyword(s):  
Angiotensin Ii ◽  
Molecular Mass ◽  
Half Life ◽  
Rna Binding ◽  
Ex Vivo ◽  
Cross Link ◽  
Band Shift ◽  
Incubation System ◽  
A Cell

ABSTRACT Several authors have shown that angiotensin II stimulates hepatic angiotensinogen synthesis in vivo, ex vivo and in vitro. In previous studies we have demonstrated that this effect of angiotensin II depends mainly on a transient inhibition of adenylyl cyclase and is the consequence of a stabilization of angiotensinogen mRNA. In the present study we describe the isolation of a polysomal 12 kDa protein which, in band shift and cross link assays, shows a specific affinity to the 3′ untranslated region (3′ UTR) of angiotensinogen mRNA and prevents enzymatic degradation of angiotensinogen mRNA in a cell-free incubation system. [32P]UTP-labelled or unlabelled 3′ fragments of angiotensinogen mRNA were synthesized on a transcription vector (pGEM5zf+) into which the corresponding DNA sequence was cloned after restriction from vector pRAG 16. Binding of the 12 kDa protein to the radioactively labelled 3′ UTR of angiotensinogen mRNA could be displaced by unlabelled 3′ UTR mRNA fragments but not by a renin mRNA of comparable length derived from the coding region, The RNA-binding protein appears to be derived from a higher molecular mass precursor (45 kDa) which is preferentially present under reducing conditions in vitro; the active low molecular mass form is evident in the absence of reducing agents. In a cross link experiment we established that a band shift signal which was obtained in the presence of the 45 kDa protein preparation exclusively depends on RNA binding of the active 12 kDa protein. In addition, a phosphorylation step may be involved in the activation of the 12 kDa protein, since its molecular mass and isoelectric point correlate with proteins which were phosphorylated in response to transient decreases of cAMP (induced by guanfacine or angiotensin II) or in response to a direct inhibition of protein kinase A by the cAMP antagonist Rp-cAMP. The importance of phosphorylation reactions for the stabilization of angiotensinogen mRNA was further assessed in a cell-free incubation system of rat liver parenchymal cells. These studies demonstrated that in the presence of acid phosphatase (1 U/ml) the half-life of angiotensinogen was significantly decreased. In the same incubation system the 12 kDa protein increased the half-life of endogenous as well as of exogenous angiotensinogen mRNA three- to fourfold, while no stabilizing effect was apparent when exogenous angiotensinogen mRNA from which the 3′ tail had been deleted was added. We concluded that an intracellular 12 kDa protein may play a crucial role in the angiotensin Il-induced stabilization of hepatic angiotensinogen mRNA and further suggest that this protein exerts its effect via binding to the 3′ UTR of angiotensinogen mRNA in response to a cAMP-dependent activation step.

scholarly journals Ifit2 Is a Restriction Factor in Rabies Virus Pathogenicity

2017 ◽  
Vol 91 (17) ◽  
Author(s):  
Benjamin M. Davis ◽  
Volker Fensterl ◽  
Tessa M. Lawrence ◽  
Andrew W. Hudacek ◽  
Ganes C. Sen ◽  
...  
Keyword(s):  
Viral Replication ◽  
Rabies Virus ◽  
Rna Binding ◽  
Binding Capacity ◽  
Severe Disease ◽  
Case Fatality ◽  
Brain Regions ◽  
Restriction Factor

ABSTRACT Understanding the interactions between rabies virus (RABV) and individual host cell proteins is critical for the development of targeted therapies. Here we report that interferon-induced protein with tetratricopeptide repeats 2 (Ifit2), an interferon-stimulated gene (ISG) with possible RNA-binding capacity, is an important restriction factor for rabies virus. When Ifit2 was depleted, RABV grew more quickly in mouse neuroblastoma cells in vitro. This effect was replicated in vivo, where Ifit2 knockout mice displayed a dramatically more severe disease phenotype than wild-type mice after intranasal inoculation of RABV. This increase in pathogenicity correlated to an increase in RABV mRNA and live viral load in the brain, as well as to an accelerated spread to brain regions normally affected by this RABV model. These results suggest that Ifit2 exerts its antiviral effect mainly at the level of viral replication, as opposed to functioning as a mechanism that restricts viral entry/egress or transports RABV particles through axons. IMPORTANCE Rabies is a fatal zoonotic disease with a nearly 100% case fatality rate. Although there are effective vaccines for rabies, this disease still takes the lives of about 50,000 people each year. Victims tend to be children living in regions without comprehensive medical infrastructure who present to health care workers too late for postexposure prophylaxis. The protein discussed in our report, Ifit2, is found to be an important restriction factor for rabies virus, acting directly or indirectly against viral replication. A more nuanced understanding of this interaction may reveal a step of a pathway or site at which the system could be exploited for the development of a targeted therapy.

scholarly journals Human La protein: a stabilizer of histone mRNA.

1997 ◽  
Vol 17 (6) ◽  
pp. 3028-3036 ◽  
Author(s):  
R S McLaren ◽  
N Caruccio ◽  
J Ross
Keyword(s):  
Half Life ◽  
Mrna Decay ◽  
Rna Binding ◽  
S Phase ◽  
Coding Region ◽  
Histone Mrna ◽  
Histone Proteins ◽  
Unbound Fraction ◽  
Mrna Half Life

Histone mRNA is destabilized at the end of S phase and in cell-free mRNA decay reaction mixtures supplemented with histone proteins, indicating that histones might autoregulate the histone mRNA half-life. Histone mRNA destabilization in vitro requires three components: polysomes, histones, and postpolysomal supernatant (S130). Polysomes are the source of the mRNA and mRNA-degrading enzymes. To investigate the role of the S130 in autoregulation, crude S130 was fractionated by histone-agarose affinity chromatography. Two separate activities affecting the histone mRNA half-life were detected. The histone-agarose-bound fraction contained a histone mRNA destabilizer that was activated by histone proteins; the unbound fraction contained a histone mRNA stabilizer. Further chromatographic fractionation of unbound material revealed only a single protein stabilizer, which was purified to homogeneity, partially sequenced, and found to be La, a well-characterized RNA-binding protein. When purified La was added to reaction mixtures containing polysomes, a histone mRNA decay intermediate was stabilized. This intermediate corresponded to histone mRNA lacking 12 nucleotides from its 3' end and containing an intact coding region. Anti-La antibody blocked the stabilization effect. La had little or no effect on several other cell cycle-regulated mRNAs. We suggest that La prolongs the histone mRNA half-life during S phase and thereby increases histone protein production.

scholarly journals All Three Domains of the Hepatitis C Virus Nonstructural NS5A Protein Contribute to RNA Binding

2010 ◽  
Vol 84 (18) ◽  
pp. 9267-9277 ◽  
Author(s):  
Toshana L. Foster ◽  
Tamara Belyaeva ◽  
Nicola J. Stonehouse ◽  
Arwen R. Pearson ◽  
Mark Harris
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Binding ◽  
Nonstructural Protein ◽  
Binding Capacity ◽  
Specific Interaction ◽  
Genome Replication ◽  
Mobility Shift ◽  
Positive Strand Rna

ABSTRACT The hepatitis C virus (HCV) nonstructural protein NS5A is critical for viral genome replication and is thought to interact directly with both the RNA-dependent RNA polymerase, NS5B, and viral RNA. NS5A consists of three domains which have, as yet, undefined roles in viral replication and assembly. In order to define the regions that mediate the interaction with RNA, specifically the HCV 3′ untranslated region (UTR) positive-strand RNA, constructs of different domain combinations were cloned, bacterially expressed, and purified to homogeneity. Each of these purified proteins was probed for its ability to interact with the 3′ UTR RNA using filter binding and gel electrophoretic mobility shift assays, revealing differences in their RNA binding efficiencies and affinities. A specific interaction between domains I and II of NS5A and the 3′ UTR RNA was identified, suggesting that these are the RNA binding domains of NS5A. Domain III showed low in vitro RNA binding capacity. Filter binding and competition analyses identified differences between NS5A and NS5B in their specificities for defined regions of the 3′ UTR. The preference of NS5A, in contrast to NS5B, for the polypyrimidine tract highlights an aspect of 3′ UTR RNA recognition by NS5A which may play a role in the control or enhancement of HCV genome replication.

Characterization of an insect ferritin subunit synthesized in a cell-free system

1990 ◽  
Vol 68 (7-8) ◽  
pp. 1005-1011 ◽  
Author(s):  
C. A. Ketola-Pirie
Keyword(s):  
Molecular Mass ◽  
Proteinase K ◽  
Translation Product ◽  
Western Blots ◽  
Free System ◽  
Cell Free System ◽  
A Cell ◽  
Vacuolar System

Ferritin, an iron-sequestering and -binding protein, is localized to the vacuolar system in Calpodes ethlius larvae. The amount of iron-loaded ferritin in intact larval midgut can be increased by pretreatment with iron. When poly(A)+ RNA from control or iron-treated larvae was translated in vitro, a 24 kilodalton (kDa) protein was a major translation product. If the cell-free system was supplemented with dog pancreatic microsomes, the 24-kDa protein was not detectable: the major translation product was 28–30 kDa. The 24-kDa and 28- to 30-kDa proteins were identified as ferritin subunits by immunoprecipitation with anti-Manduca ferritin antibodies. Proteinase K digestion of the translation products showed that the 28- to 30-kDa subunit was targeted into the lumen of, and protected by, the microsomes. The change in molecular mass of the ferritin monomer was attributed to glycosylation of the 24-kDa subunit within the lumen of the microsomes. This was demonstrated by (i) the ability of the 28- to 30-kDa subunit, but not the 24-kDa subunit, to bind concanavalin A on Western blots and (ii) inhibition of the change in molecular mass from 24 to 28–30 kDa if tunicamycin is added to the microsomes. The results indicate that the Calpodes ferritin subunit was synthesized, targeted to microsomes, and glycosylated within their lumen in a rabbit reticulocyte cell-free system primed with midgut poly(A)+ RNA extracted from control or iron-treated larvae.Key words: insect ferritin, cell-free synthesis, glycosylation.

scholarly journals Regulatory Interactions of Csr Components: the RNA Binding Protein CsrA Activates csrB Transcription inEscherichia coli

2001 ◽  
Vol 183 (20) ◽  
pp. 6017-6027 ◽  
Author(s):  
Seshagirirao Gudapaty ◽  
Kazushi Suzuki ◽  
Xin Wang ◽  
Paul Babitzke ◽  
Tony Romeo
Keyword(s):  
Rna Binding ◽  
Binding Capacity ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Growth Curves ◽  
Regulatory Interactions ◽  
Transcript Stability ◽  
Carbon Storage Regulator

ABSTRACT The global regulator CsrA (carbon storage regulator) ofEscherichia coli is a small RNA binding protein that represses various metabolic pathways and processes that are induced in the stationary phase of growth, while it activates certain exponential phase functions. Both repression and activation by CsrA involve posttranscriptional mechanisms, in which CsrA binding to mRNA leads to decreased or increased transcript stability, respectively. CsrA also binds to a small untranslated RNA, CsrB, forming a ribonucleoprotein complex, which antagonizes CsrA activity. We have further examined the regulatory interactions of CsrA and CsrB RNA. The 5′ end of the CsrB transcript was mapped, and acsrB::cam null mutant was constructed. CsrA protein and CsrB RNA levels were estimated throughout the growth curves of wild-type and isogenic csrA,csrB, rpoS, or csrA rpoSmutant strains. CsrA levels exhibited modest or negligible effects of these mutations. The intracellular concentration of CsrA exceeded the total CsrA-binding capacity of intracellular CsrB RNA. In contrast, CsrB levels were drastically decreased (∼10-fold) in thecsrA mutants. CsrB transcript stability was unaffected by csrA. The expression of a csrB-lacZtranscriptional fusion containing the region from −242 to +4 bp of thecsrB gene was decreased ∼20-fold by acsrA::kanR mutation in vivo but was unaffected by CsrA protein in vitro. These results reveal a significant, though most likely indirect, role for CsrA in regulatingcsrB transcription. Furthermore, our findings suggest that CsrA mediates an intriguing form of autoregulation, whereby its activity, but not its levels, is modulated through effects on an RNA antagonist, CsrB.

scholarly journals Production of Membrane Vesicles in Listeria monocytogenes Cultured with or without Sub-Inhibitory Concentrations of Antibiotics and Their Innate Immune Responses In Vitro

Genes ◽  
2021 ◽  
Vol 12 (3) ◽  
pp. 415
Author(s):  
Jung-Hwa Woo ◽  
Shukho Kim ◽  
Taewon Lee ◽  
Je-Chul Lee ◽  
Ji-Hyun Shin
Keyword(s):  
Listeria Monocytogenes ◽  
Inflammatory Responses ◽  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Host Cells ◽  
Proteinase K ◽  
Tnf Α ◽  
Food Borne Illness ◽  
Proteinase K Treatment

Listeriosis is a food-borne illness caused by Listeria monocytogenes. Ampicillin (AMP) alone or in combination with gentamicin (GEN) is the first-line treatment option. Membrane vesicle (MV) production in L. monocytogenes under antibiotic stress conditions and pathologic roles of these MVs in hosts have not been reported yet. Thus, the aim of this study was to investigate the production of MVs in L. monocytogenes cultured with sub-minimum inhibitory concentrations (MICs) of AMP, GEN, or trimethoprim/sulfamethoxazole (SXT) and determine pathologic effects of these MVs in colon epithelial Caco-2 cells. L. monocytogenes cultured in tryptic soy broth with 1/2 MIC of AMP, GEN, or SXT produced 6.0, 2.9, or 1.5 times more MV particles, respectively, than bacteria cultured without antibiotics. MVs from L. monocytogenes cultured with AMP (MVAMP), GEN (MVGEN), or SXT (MVSXT) were more cytotoxic to Caco-2 cell than MVs obtained from cultivation without antibiotics (MVTSB). MVAMP induced more expression of tumor necrosis factor (TNF)-α gene than MVTSB, MVGEN and MVSXT, whereas MVTSB induced more expression of interleukin (IL)-1β and IL-8 genes than other MVs. Expression of pro-inflammatory cytokine genes by L. monocytogenes MVs was significantly inhibited by proteinase K treatment of MVs. In conclusion, antibiotic stress can trigger the biogenesis of MVs in L. monocytogenes and MVs produced by L. monocytogenes exposed to sub-MIC of AMP can induce strong pro-inflammatory responses by expressing TNF-α gene in host cells, which may contribute to the pathology of listeriosis.

scholarly journals Angptl2 is a Marker of Cellular Senescence: The Physiological and Pathophysiological Impact of Angptl2-Related Senescence

2021 ◽  
Vol 22 (22) ◽  
pp. 12232
Author(s):  
Nathalie Thorin-Trescases ◽  
Pauline Labbé ◽  
Pauline Mury ◽  
Mélanie Lambert ◽  
Eric Thorin
Keyword(s):  
Cell Fate ◽  
Cellular Senescence ◽  
Cellular Response ◽  
Inflammatory Diseases ◽  
Age Related ◽  
A Cell ◽  
Future Work ◽  
The Impact

Cellular senescence is a cell fate primarily induced by DNA damage, characterized by irreversible growth arrest in an attempt to stop the damage. Senescence is a cellular response to a stressor and is observed with aging, but also during wound healing and in embryogenic developmental processes. Senescent cells are metabolically active and secrete a multitude of molecules gathered in the senescence-associated secretory phenotype (SASP). The SASP includes inflammatory cytokines, chemokines, growth factors and metalloproteinases, with autocrine and paracrine activities. Among hundreds of molecules, angiopoietin-like 2 (angptl2) is an interesting, although understudied, SASP member identified in various types of senescent cells. Angptl2 is a circulatory protein, and plasma angptl2 levels increase with age and with various chronic inflammatory diseases such as cancer, atherosclerosis, diabetes, heart failure and a multitude of age-related diseases. In this review, we will examine in which context angptl2 was identified as a SASP factor, describe the experimental evidence showing that angptl2 is a marker of senescence in vitro and in vivo, and discuss the impact of angptl2-related senescence in both physiological and pathological conditions. Future work is needed to demonstrate whether the senescence marker angptl2 is a potential clinical biomarker of age-related diseases.

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