Maintenance of protein synthesis in spite of mRNA breakdown in interferon-treated HeLa cells infected with reovirus

1983 ◽  
Vol 3 (1) ◽  
pp. 64-69
Author(s):  
T W Nilsen ◽  
P A Maroney ◽  
C Baglioni
Keyword(s):  
Protein Synthesis ◽  
Hela Cells ◽  
Large Fraction ◽  
Relative Concentration ◽  
Mrna Degradation ◽  
Rnase L ◽  
Mrna Synthesis ◽  
Double Stranded Rna ◽  
Specific Mrnas ◽  
Cloned Cdna

Interferon induces the synthesis of an enzyme which synthesizes 2',5'-oligoadenylate [2',5'-oligo(A)] when activated by double-stranded RNA. The 2',5'-oligo(A) in turn activates an endonuclease (RNase L). Concentrations of 2',5'-oligo(A) sufficient to activate RNase L are formed in interferon-treated HeLa cells infected with reovirus, and a large fraction of cellular mRNA is degraded (T. W. Nilsen, P. A. Maroney, and C. Baglioni, J. Virol. 42:1039-1045, 1982). We report here that in spite of this mRNA degradation, protein synthesis was not significantly inhibited in these cells. When mRNA synthesis was inhibited with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, protein synthesis was markedly decreased, as shown by reduced incorporation of labeled amino acids and a decrease in polyribosomes. This suggested that the turnover of mRNA could be compensated for by increased production of mRNA. The relative concentration of specific mRNAs was measured with cloned cDNA probes. The amount of these mRNAs present in control cells was comparable to that in interferon-treated cells infected with reovirus, whereas it was decreased in the latter cells treated with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole.

scholarly journals Maintenance of protein synthesis in spite of mRNA breakdown in interferon-treated HeLa cells infected with reovirus.

1983 ◽  
Vol 3 (1) ◽  
pp. 64-69 ◽  
Author(s):  
T W Nilsen ◽  
P A Maroney ◽  
C Baglioni
Keyword(s):  
Protein Synthesis ◽  
Hela Cells ◽  
Large Fraction ◽  
Relative Concentration ◽  
Mrna Degradation ◽  
Rnase L ◽  
Mrna Synthesis ◽  
Double Stranded Rna ◽  
Specific Mrnas ◽  
Cloned Cdna

Interferon induces the synthesis of an enzyme which synthesizes 2',5'-oligoadenylate [2',5'-oligo(A)] when activated by double-stranded RNA. The 2',5'-oligo(A) in turn activates an endonuclease (RNase L). Concentrations of 2',5'-oligo(A) sufficient to activate RNase L are formed in interferon-treated HeLa cells infected with reovirus, and a large fraction of cellular mRNA is degraded (T. W. Nilsen, P. A. Maroney, and C. Baglioni, J. Virol. 42:1039-1045, 1982). We report here that in spite of this mRNA degradation, protein synthesis was not significantly inhibited in these cells. When mRNA synthesis was inhibited with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, protein synthesis was markedly decreased, as shown by reduced incorporation of labeled amino acids and a decrease in polyribosomes. This suggested that the turnover of mRNA could be compensated for by increased production of mRNA. The relative concentration of specific mRNAs was measured with cloned cDNA probes. The amount of these mRNAs present in control cells was comparable to that in interferon-treated cells infected with reovirus, whereas it was decreased in the latter cells treated with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole.

scholarly journals Activation of p38 Mitogen-Activated Protein Kinase and c-Jun NH2-Terminal Kinase by Double-Stranded RNA and Encephalomyocarditis Virus: Involvement of RNase L, Protein Kinase R, and Alternative Pathways

2000 ◽  
Vol 20 (2) ◽  
pp. 617-627 ◽  
Author(s):  
Mihail S. Iordanov ◽  
Jayashree M. Paranjape ◽  
Aimin Zhou ◽  
John Wong ◽  
Bryan R. G. Williams ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Protein Kinase ◽  
P38 Mapk ◽  
Encephalomyocarditis Virus ◽  
Mitogen Activated Protein Kinase ◽  
Rnase L ◽  
Double Stranded Rna ◽  
Mitogen Activated Protein ◽  
Stimulation Of ◽  
In Cells

ABSTRACT Double-stranded RNA (dsRNA) accumulates in virus-infected mammalian cells and signals the activation of host defense pathways of the interferon system. We describe here a novel form of dsRNA-triggered signaling that leads to the stimulation of the p38 mitogen-activated protein kinase (p38 MAPK) and the c-Jun NH2-terminal kinase (JNK) and of their respective activators MKK3/6 and SEK1/MKK4. The dsRNA-dependent signaling to p38 MAPK was largely intact in cells lacking both RNase L and the dsRNA-activated protein kinase (PKR), i.e., the two best-characterized mediators of dsRNA-triggered antiviral responses. In contrast, activation of both MKK4 and JNK by dsRNA was greatly reduced in cells lacking RNase L (or lacking both RNase L and PKR) but was restored in these cells when introduction of dsRNA was followed by inhibition of ongoing protein synthesis or transcription. These results are consistent with the notion that the role of RNase L and PKR in the activation of MKK4 and JNK is the elimination, via inhibition of protein synthesis, of a labile negative regulator(s) of the signaling to JNK acting upstream of SEK1/MKK4. In the course of these studies, we identified a long-sought site of RNase L-mediated cleavage in the 28S rRNA, which could cause inhibition of translation, thus allowing the activation of JNK by dsRNA. We propose that p38 MAPK is a general participant in dsRNA-triggered cellular responses, whereas the activation of JNK might be restricted to cells with reduced rates of protein synthesis. Our studies demonstrate the existence of alternative (RNase L- and PKR-independent) dsRNA-triggered signaling pathways that lead to the stimulation of stress-activated MAPKs. Activation of p38 MAPK (but not of JNK) was demonstrated in mouse fibroblasts in response to infection with encephalomyocarditis virus (ECMV), a picornavirus that replicates through a dsRNA intermediate. Fibroblasts infected with EMCV (or treated with dsRNA) produced interleukin-6, an inflammatory and pyrogenic cytokine, in a p38 MAPK-dependent fashion. These findings suggest that stress-activated MAPKs participate in mediating inflammatory and febrile responses to viral infections.

scholarly journals Real-time 2-5A kinetics suggest that interferons β and λ evade global arrest of translation by RNase L

2019 ◽  
Vol 116 (6) ◽  
pp. 2103-2111 ◽  
Author(s):  
Alisha Chitrakar ◽  
Sneha Rath ◽  
Jesse Donovan ◽  
Kaitlin Demarest ◽  
Yize Li ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Innate Immune System ◽  
Innate Immune ◽  
Host Protein ◽  
Rna Cleavage ◽  
Rnase L ◽  
Foreign Material ◽  
Defense Proteins ◽  
Double Stranded Rna ◽  
Host Protein Synthesis

Cells of all mammals recognize double-stranded RNA (dsRNA) as a foreign material. In response, they release interferons (IFNs) and activate a ubiquitously expressed pseudokinase/endoribonuclease RNase L. RNase L executes regulated RNA decay and halts global translation. Here, we developed a biosensor for 2′,5′-oligoadenylate (2-5A), the natural activator of RNase L. Using this biosensor, we found that 2-5A was acutely synthesized by cells in response to dsRNA sensing, which immediately triggered cellular RNA cleavage by RNase L and arrested host protein synthesis. However, translation-arrested cells still transcribed IFN-stimulated genes and secreted IFNs of types I and III (IFN-β and IFN-λ). Our data suggest that IFNs escape from the action of RNase L on translation. We propose that the 2-5A/RNase L pathway serves to rapidly and accurately suppress basal protein synthesis, preserving privileged production of defense proteins of the innate immune system.

scholarly journals RNase L reprograms translation by widespread mRNA turnover escaped by antiviral mRNAs

2018 ◽  
Author(s):  
James M Burke ◽  
Stephanie L Moon ◽  
Evan T Lester ◽  
Tyler Matheny ◽  
Roy Parker
Keyword(s):  
Translation Initiation ◽  
Mammalian Cells ◽  
Mrna Degradation ◽  
Stress Granule ◽  
Mrna Turnover ◽  
Rnase L ◽  
Protein Kinase R ◽  
Double Stranded Rna ◽  
Translation Repression ◽  
Eif2α Phosphorylation

SUMMARYIn response to foreign and endogenous double-stranded RNA (dsRNA), protein kinase R (PKR) and ribonuclease L (RNase L) reprogram translation in mammalian cells. PKR inhibits translation initiation through eIF2α phosphorylation, which triggers stress granule (SG) formation and promotes translation of stress responsive mRNAs. The mechanisms of RNase L-driven translation repression, its contribution to SG assembly, and its regulation of dsRNA stress-induced mRNAs are unknown. We demonstrate that RNase L drives translational shut-off in response to dsRNA by promoting widespread turnover of mRNAs. This alters stress granule assembly and reprograms translation by only allowing for the translation of mRNAs resistant to RNase L degradation, including numerous antiviral mRNAs such asIFN-β. Individual cells differentially activate dsRNA responses revealing variation that can affect cellular outcomes. This identifies bulk mRNA degradation and the resistance of antiviral mRNAs as the mechanism by which RNaseL reprograms translation in response to dsRNA.

scholarly journals Concerted 2-5A-Mediated mRNA Decay and Transcription Reprogram Protein Synthesis in dsRNA Response

2018 ◽  
Author(s):  
Sneha Rath ◽  
Eliza Prangley ◽  
Jesse Donovan ◽  
Kaitlin Demarest ◽  
Yigal Meir ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Translation Initiation ◽  
Mrna Stability ◽  
Rna Degradation ◽  
Innate Immune ◽  
Messenger Rnas ◽  
Rnase L ◽  
Rna Seq ◽  
Defense Proteins ◽  
Double Stranded Rna

RNA degradation by RNase L during 2-5A-mediated decay (2-5AMD) is a conserved mammalian stress response to viral and endogenous double-stranded RNA (dsRNA). 2-5AMD onsets rapidly and facilitates a switch of protein synthesis from homeostasis to production of interferons (IFNs). To understand the mechanism of this protein synthesis reprogramming, we examined 2-5AMD in human cells. 2-5AMD triggers polysome collapse characteristic of a translation initiation defect, but translation initiation complexes and ribosomes purified from the translation-arrested cells remain functional. Using spike-in RNA-seq we found that basal messenger RNAs (mRNAs) rapidly decay, while mRNAs encoding IFNs and IFN-stimulated genes evade 2-5AMD and accumulate. The IFN evasion results from the combined effect of better mRNA stability and positive feedback amplification in the IFN response. Therefore, 2-5AMD and transcription act in concert to revamp the cellular mRNA composition. The resulting preferential accumulation of innate immune mRNAs establishes 'prioritized' synthesis of defense proteins.

scholarly journals A GM-colony-stimulating factor (CSF) activated ribonuclease system transregulates M-CSF receptor expression in the murine FDC-P1/MAC myeloid cell line.

1992 ◽  
Vol 3 (5) ◽  
pp. 535-544 ◽  
Author(s):  
B C Gliniak ◽  
L S Park ◽  
L R Rohrschneider
Keyword(s):  
Protein Synthesis ◽  
Cell Line ◽  
Transcriptional Activation ◽  
Mrna Degradation ◽  
Receptor Expression ◽  
Metabolic Inhibitors ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Stimulating Factor

The murine myeloid precursor cell line FDC-P1/MAC simultaneously expresses receptors for multi-colony-stimulating factor (CSF), granulocyte-macrophage (GM)-CSF, and macrophage (M)-CSF. Growth of FDC-P1/MAC cells in either multi-CSF or GM-CSF results in the posttranscriptional suppression of M-CSF receptor (c-fms proto-oncogene) expression. We use the term transregulation to describe this control of receptor expression and have further characterized this regulatory process. The removal of FDC-P1/MAC cells from GM-CSF stimulation resulted in the re-expression of c-fms mRNA independent of M-CSF stimulation and new protein synthesis. Switching FDC-P1/MAC cells from growth in M-CSF to GM-CSF caused the selective degradation of c-fms mRNA within 6 h after factor switching. Blocking protein synthesis or gene transcription with metabolic inhibitors effectively prevented GM-CSF stimulated degradation of c-fms mRNA. These results suggest that the transregulation of c-fms transcripts by GM-CSF requires the transcriptional activation of a selective mRNA degradation factor. In vitro analysis, the use of cytoplasmic cell extracts, provided evidence that a ribonuclease is preferentially active in GM-CSF stimulated cells, although the specificity for mRNA degradation in vitro is broader than seen in vivo. Together, these data suggest that GM-CSF can dominantly transregulate the level of c-fms transcript through the transcriptional activation of a ribonuclease degradation system.

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