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

Abstract

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. 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 secretedIFNs of types I and III (IFN-β and IFN-). 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.. CC-BY 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/476341 doi: bioRxiv preprintThe 2-5A system is also a surveillance pathway for endogenous dsRNAs from mammalian genomes. Cells with adenosine deaminase 1 (ADAR1) deficiency accumulate self-dsRNA that promotes 2-5A-driven apoptosis 8 . 2-5A synthesis in the presence of low amounts of endogenous dsRNAs does not cause cell death, but functions as a suppressor of adhesion, proliferation, migration, and prostate cancer metastasis 17,18 . Additionally, activation of the 2-5A system also blocks secretion of milk . CC-BY 4.0 International license is made available under a

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

“…The induction of defense transcripts bypasses RNase L ( Fig. 6D; S6A; S7A), as previously reported (Alisha Chitrakar et al, 2018). To test whether defense mRNAs could survive in the presence of RNase L, we measured their decay kinetics during 2-5AMD in the presence of actinomycin D treatment, which was used to stop new transcription ( Fig.…”

“…During homeostasis, 2-5AMD regulates adhesion and migration activity of mammalian cells (Banerjee et al, 2015;. Upon acute activation by dsRNA, 2-5AMD arrests global translation by an immediate and poorly understood mechanism (Alisha Chitrakar et al, 2018;. In mammalian cells, dsRNA inhibits translation not only via 2-5AMD, but also via phosphorylation of the translation initiation factor eIF2 by the serine-threonine kinase protein kinase R (PKR) (Fig.…”

“…Due to the complexity of the RNA degradation program, the arrest of global translation by 2-5AMD could not be linked to any specific RNA (Alisha Chitrakar et al, 2018;.…”

“…Shortly after activation of 2-5AMD, dsRNA triggers the transcriptional IFN response, leading to upregulation of innate immune mRNAs ( Fig. 1A) (Alisha Chitrakar et al, 2018;Kawai et al, 2005;Liu et al, 2008). While global translation remains silenced by actively ongoing 2-5AMD, the mRNAs encoding the interferons IFN-β (type I) and IFN- (type III) bypass the global inhibition and are actively translated (Alisha Chitrakar et al, 2018).…”

“…1A) (Alisha Chitrakar et al, 2018;Kawai et al, 2005;Liu et al, 2008). While global translation remains silenced by actively ongoing 2-5AMD, the mRNAs encoding the interferons IFN-β (type I) and IFN- (type III) bypass the global inhibition and are actively translated (Alisha Chitrakar et al, 2018). Here we employ cell biology, proteomics, transcriptomics and modeling approaches to determine the translation reprogramming mechanism of 2-5AMD and to explain how innate immune mRNAs can evade the translational arrest.…”