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

Moon, Stephanie L.; Lester, Evan; Matheny, Tyler; Parker, Roy

doi:10.1101/486530

Cited by 10 publications

(19 citation statements)

References 63 publications

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“…Thus, DENV RNA is largely unaffected by RNase L-mediated mRNA decay. Consequently, this permits DENV protein production, consistent with cytoplasmic mRNAs having the capacity to be translated during the RNase L response (9,10).…”

Section: Resultsmentioning

confidence: 92%

“…Dengue virus mRNAs escape RNase L-mediated mRNA decay and produce protein DENV is a flavivirus (+ssRNA virus) that replicates in the cytoplasm. To investigate the mechanism by which DENV evades the effects of RNase L (8), we infected parental (WT) and RNase L-KO (RL-KO) A549 cells with dengue virus serotype 2 and performed single-molecule fluorescent in situ hybridization (smFISH) for DENV mRNA, GAPDH mRNA, which is degraded when RNase L is active (9), and also stained for DENV NS3 protein production by immunofluorescence.…”

Section: Resultsmentioning

confidence: 99%

“…Second, rapid and widespread decay of host mRNAs by RNase L accounts for RNase L-mediated reduction in translation (9,10). In light of these findings, it is unclear how RNase L limits viral protein synthesis, how some viruses escape the effects of RNase L, and how RNase L activation affects host antiviral protein production.…”

Section: Introductionmentioning

confidence: 99%

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RNase L limits host and viral protein synthesis via inhibition of mRNA export

Gilchrist

Sawyer

Parker

2021

Preprint

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RNase L is widely thought to limit viral protein synthesis by cleaving host rRNA and viral mRNA, resulting in translation arrest and viral mRNA degradation. Herein, we show that the mRNAs of dengue virus and influenza A virus largely escape RNase L-mediated mRNA decay, and this permits viral protein production. However, activation of RNase L arrests nuclear mRNA export, which strongly inhibits influenza A virus protein synthesis and reduces cytokine production. Importantly, the heterogeneous and temporal nature of the mRNA export block in individual cells permits sufficient production of antiviral cytokines from transcriptionally induced host mRNAs. This defines RNase L-mediated arrest of mRNA export as a key antiviral shutoff and cytokine regulatory pathway.

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Section: Resultsmentioning

confidence: 92%

Section: Resultsmentioning

confidence: 99%

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RNase L limits host and viral protein synthesis via inhibition of mRNA export

Gilchrist

Sawyer

Parker

2021

Preprint

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“…As previously described (Burke et al, 2019), HEK293T cells (T25 Flask at 80% confluence) were co-transfected with 1ug of pLenti-SRSF2-mCherry-blasticydin, 1ug of pVSV-G, 1ug of pRSV-Rev, and 1ug of pMDLg-pRRe using 16uL of lipofectamine 2000. Medium was replaced 6 hours post-transfection.…”

Section: Generation Of Lentiviral Particlesmentioning

confidence: 99%

Tau aggregates are RNA-protein assemblies that mis-localize multiple nuclear speckle components

Lester

Ooi

Bakkar

et al. 2021

Preprint

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Tau aggregates contribute to neurodegenerative diseases including frontotemporal dementia and Alzheimer’s disease (AD). Although RNA promotes tau aggregation in vitro, whether tau aggregates in cells contain RNA is unknown. We demonstrate in cell culture and mouse brains that both cytosolic and nuclear tau aggregates contain RNA, with enrichment for snRNAs and snoRNAs. Nuclear tau aggregates colocalize with and alter the composition, dynamics, and organization of nuclear speckles, which are membraneless organelles involved in pre-mRNA splicing. Moreover, several nuclear speckle components, including SRRM2, mislocalize to cytosolic tau aggregates in cells, mouse brains, and patient brains with AD, frontotemporal dementia (FTD), and corticobasal degeneration (CBD). Consistent with these alterations we observe the presence of tau aggregates is sufficient to alter pre-mRNA splicing. This work identifies tau alteration of nuclear speckles as a feature of tau aggregation that may contribute to the pathology of tau aggregates.

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“…The proposed mechanisms that cells may utilize to restrict the formation or disassembly SGs/ MOs involve regulation of RNA synthesis rates and RNA modifications underlying RNA-RNA interactions, ribosome association and regulation of RNA concentrations through RNA decay pathway (Burke et al, 2019. RNA sensing is central to viral infections, therefore, RNA unwinding enzymes called RNA helicases (DEAD-box proteins) can perturb RNA-RNA interactions by invoking their inherent ATPase activity.…”

Section: Extending the Interferon-ribosomal-rna Pathway Axis To Coagumentioning

confidence: 99%

Proteomic Profiling of Protease-Primed Virus-Permissive Caco-2 Cells Display Abortive-Interferon Pathway and Deregulated Thromboinflammatory SERPINS

Sharma¹,

Panda²

2020

Preprint

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Emerging paradigms in interferon (IFN) biology suggest a dynamic INF induced interactome that extends through broader Interferon Stimulated Gene (ISG)- induction, which implicates interferon- ISG coordinated cross-talk with mRNA processing, post-translational modification and metabolic processes that underlie pathological (viral, autoimmune and tumor biology) and physiological (stem cell regenerative pathways) processes. INF immune responses can also be triggered by endogenous host-derived molecules that are generated in response to cellular stress or hemostasis imbalance to establish tissue repair and regeneration in first place, however, overactivation or lack of countermeasures can result in host tissue damage. The proteases are integral to viral and tumor pathology, and importantly serine proteases TMPRSS2 and trypsin have been identified as important molecular determinants underlying COVID-19 pathology, and emergence of coronaviruses cultured in vitro, respectively. We propose that pathogen associated proteases can act as novel stress-inducers to facilitate viral- competent immunomodulation. We term it as Protease Induced Transcriptomic/ epi-Transcriptomic Reshaping (PITTR) of host cells to counter cellular stress. We present a novel experimental model and our preliminary findings of trypsin- primed Caco-2 cells (CPT) that result in translational halt comparable to cells grown under serum-starvation conditions (CSS). CPT at escalating trypsin concentration (CPT- EC) induce upregulation of selective proteins that majorly map to ribosomal, RNA transport, and spliceosome ribonucleoproteins (RNPs). The inclusion of proinflammatory IL1-b to CPT (CPT- IL) resulted in global overexpression of proteins comparable to Caco-2 cells cultured in growth-factor rich serum conditions (CFBS), indicating a likely de-repression of trypsin- induced translational halt. Caco-2 cells display abortive interferon proteome under differential trypsin conditions (CPT, CPT-EC and CPT-IL), which is marked by complete lack of INF generation despite induction of intermediate ISGs, suggestive of protease (trypsin)- dependent regulation of INF response. Viruses regulate the proteome of stress granules (SGs) that are induced to cope transient translational halt as a central adaptive response to pathogen induced cellular stress. The integral components of SGs include non-translating mRNA, ribonucleoproteins (RNPs) and RNA binding proteins (RBPs), which together form biological condensates through a biophysical process involving weak electrostatic interactions through intrinsically disordered regions in RBPs resulting in liquid- liquid phase separation. We compared the CPT- EC proteome to the Mammalian Stress Granules Proteome (MSGP) database to explore potential RBPs that could possibly regulate INF response (and could act as potential anti-viral targets). Notably, differentially upregulated RNPs and potential RBPs from ISG family including ADAR and PRKRA, and RNA helicases implicated in viral pathogenesis were found to be upregulated in the CPT- EC proteome further strengthening the role of proteases (trypsin) in regulating INF pathways independent of the pathogen. We propose that the supplementation of viable SARS-CoV-2 viral loads to trypsin- primed host cells could recapitulate an infectious disease model, which may closely phenocopy pathogen- driven inflammation and signaling events. Based on the global downregulation of seven SERPINS (serine protease inhibitors) linked to thromboinflammation in our LCMS profiling data, we support the candidature of serine protease inhibitors for protease mediated viral pathologies. COVID-19 is increasingly linked to coagulopathy and resemblance to Neutrophil Extracellular Trap (NET) related thromboinflammatory features; SERPIN A1AT (alpha 1 antitrypsin) being a potent neutrophil- elastase inhibitor and a negative regulator of coagulation complement pathway may be a promising candidate for establishing hemostasis rebalancing in COVID-19 pathology.

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

Cited by 10 publications

References 63 publications

RNase L limits host and viral protein synthesis via inhibition of mRNA export

RNase L limits host and viral protein synthesis via inhibition of mRNA export

Tau aggregates are RNA-protein assemblies that mis-localize multiple nuclear speckle components

Proteomic Profiling of Protease-Primed Virus-Permissive Caco-2 Cells Display Abortive-Interferon Pathway and Deregulated Thromboinflammatory SERPINS

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