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

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.

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Realtime 2-5A kinetics suggests interferons β and λ evade global arrest of translation by RNase L

10.1101/476341 ◽

2018 ◽

Cited By ~ 1

Author(s):

Alisha Chitrakar ◽

Sneha Rath ◽

Jesse Donovan ◽

Kaitlin Demarest ◽

Yize Li ◽

...

Keyword(s):

Protein Synthesis ◽

Innate Immune ◽

Host Protein ◽

Interferon Response ◽

Rnase L ◽

Defense Proteins ◽

Innate Immune Signaling ◽

Global Protein Synthesis ◽

Mammalian Enzyme ◽

Host Protein Synthesis

AbstractCells 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. 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 (ISGs) and secreted IFNs of types I and III (IFN-β and IFN-λ). Our data suggests that IFNs escape from the action of RNase L on translation. We propose that 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.SignificanceRNase L is a mammalian enzyme that can stop global protein synthesis during interferon response. Cells must balance the need to make interferons (which are proteins) with the risk to lose cell-wide translation due to RNase L. This balance can most simply be achieved if RNase L was activated late in the interferon response. However, we show by engineering a biosensor for the RNase L pathway, that on the contrary, RNase L activation precedes interferon synthesis. Further, translation of interferons evades the action of RNase L. Our data suggest that RNase L facilitates a switch of protein synthesis from homeostasis to specific needs of innate immune signaling.

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

10.1101/484675 ◽

2018 ◽

Cited By ~ 1

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.

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Y-RNA and tRNA Cleavage by RNase L Mediates Terminal dsRNA Response

10.1101/087106 ◽

2016 ◽

Cited By ~ 4

Author(s):

Jesse Donovan ◽

Sneha Rath ◽

David Kolet-Mandrikov ◽

Alexei Korennykh

Keyword(s):

Small Rnas ◽

Mammalian Cells ◽

Innate Immune ◽

Rna Cleavage ◽

Rnase L ◽

Rna Seq ◽

Danger Signal ◽

Double Stranded Rna ◽

Transfer Rnas ◽

Non Coding Rna

AbstractDouble-stranded RNA (dsRNA) is a danger signal that triggers endonucleolytic degradation of RNA inside infected and stressed mammalian cells. This mechanism inhibits growth and ultimately removes problematic cells via apoptosis. To elucidate the molecular functions of this program and understand the connection between RNA cleavage and programmed cell death, we visualized dsRNA-induced degradation of human small RNAs using RtcB ligase-assisted RNA sequencing (RtcB RNA-seq). RtcB RNA-seq revealed strong cleavage of select transfer RNAs (tRNAs) and autoantigenic Y-RNAs, and identified the innate immune receptor RNase L as the responsible endoribonuclease. RNase L cleaves the non-coding RNA (ncRNA) targets site-specifically, releasing abundant ncRNA fragments, and downregulating full-length tRNAs and Y-RNAs. The depletion of a single Y-RNA, RNY1, appears particularly important and the loss of this Y-RNA is sufficient to initiate apoptosis. Site-specific cleavage of small ncRNA by RNase L thus emerges as an important terminal step in dsRNA surveillance.

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Translational shutdown and evasion of the innate immune response by SARS-CoV-2 NSP14 protein

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2101161118 ◽

2021 ◽

Vol 118 (24) ◽

pp. e2101161118

Author(s):

Jack Chun-Chieh Hsu ◽

Maudry Laurent-Rolle ◽

Joanna B. Pawlak ◽

Craig B. Wilen ◽

Peter Cresswell

Keyword(s):

Protein Synthesis ◽

Active Sites ◽

Nonstructural Protein ◽

Innate Immune ◽

Host Protein ◽

Type I ◽

Inhibition Activity ◽

Health Crisis ◽

Translation Inhibition ◽

Host Protein Synthesis

The ongoing COVID-19 pandemic has caused an unprecedented global health crisis. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19. Subversion of host protein synthesis is a common strategy that pathogenic viruses use to replicate and propagate in their host. In this study, we show that SARS-CoV-2 is able to shut down host protein synthesis and that SARS-CoV-2 nonstructural protein NSP14 exerts this activity. We show that the translation inhibition activity of NSP14 is conserved in human coronaviruses. NSP14 is required for virus replication through contribution of its exoribonuclease (ExoN) and N7-methyltransferase (N7-MTase) activities. Mutations in the ExoN or N7-MTase active sites of SARS-CoV-2 NSP14 abolish its translation inhibition activity. In addition, we show that the formation of NSP14−NSP10 complex enhances translation inhibition executed by NSP14. Consequently, the translational shutdown by NSP14 abolishes the type I interferon (IFN-I)-dependent induction of interferon-stimulated genes (ISGs). Together, we find that SARS-CoV-2 shuts down host innate immune responses via a translation inhibitor, providing insights into the pathogenesis of SARS-CoV-2.

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Human RNase L tunes gene expression by selectively destabilizing the microRNA-regulated transcriptome

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1513034112 ◽

2015 ◽

Vol 112 (52) ◽

pp. 15916-15921 ◽

Cited By ~ 29

Author(s):

Sneha Rath ◽

Jesse Donovan ◽

Gena Whitney ◽

Alisha Chitrakar ◽

Wei Wang ◽

...

Keyword(s):

Gene Expression ◽

Innate Immune System ◽

Mammalian Cells ◽

Innate Immune ◽

Rnase L ◽

Double Stranded Rna ◽

Rna Targets ◽

Human Rnase ◽

Cell Growth And Differentiation ◽

Cell Adhesion And Proliferation

Double-stranded RNA (dsRNA) activates the innate immune system of mammalian cells and triggers intracellular RNA decay by the pseudokinase and endoribonuclease RNase L. RNase L protects from pathogens and regulates cell growth and differentiation by destabilizing largely unknown mammalian RNA targets. We developed an approach for transcriptome-wide profiling of RNase L activity in human cells and identified hundreds of direct RNA targets and nontargets. We show that this RNase L-dependent decay selectively affects transcripts regulated by microRNA (miR)-17/miR-29/miR-200 and other miRs that function as suppressors of mammalian cell adhesion and proliferation. RNase L mimics the effects of these miRs and acts as a suppressor of proliferation and adhesion in mammalian cells. Our data suggest that RNase L-dependent decay serves to establish an antiproliferative state via destabilization of the miR-regulated transcriptome.

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Curcumin, a Natural Antioxidant, Acts as a Noncompetitive Inhibitor of Human RNase L in Presence of Its Cofactor 2-5AIn Vitro

BioMed Research International ◽

10.1155/2014/817024 ◽

2014 ◽

Vol 2014 ◽

pp. 1-9 ◽

Cited By ~ 3

Author(s):

Ankush Gupta ◽

Pramod C. Rath

Keyword(s):

Innate Immune System ◽

Mammalian Cells ◽

Viral Infections ◽

Innate Immune ◽

Therapeutic Interventions ◽

Active Principle ◽

Rnase L ◽

Double Stranded Rna ◽

Human Rnase ◽

Noncompetitive Inhibitor

Ribonuclease L (RNase L) is an antiviral endoribonuclease of the innate immune system, which is induced and activated by viral infections, interferons, and double stranded RNA (dsRNA) in mammalian cells. Although, RNase L is generally protective against viral infections, abnormal RNase L expression and activity have been associated with a number of diseases. Here, we show that curcumin, a natural plant-derived anti-inflammatory active principle, inhibits RNase L activity; hence, it may be exploited for therapeutic interventions in case of pathological situations associated with excess activation of RNase L.

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