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

Abstract

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.

RNase L Amplifies Interferon Signaling by Inducing Protein Kinase R-Mediated Antiviral Stress Granules

“…above. Furthermore, response to polyI:C varies in cell-type dependent manner, levels of OAS isoforms as well as abundance of polyI:C-binding proteins in cells(69).Recent on September 3, 2020 by guest http://jvi.asm.org/ Downloaded from reports show the role of RNase L in widespread mRNA degradation and translation repression of select basal mRNAs while antiviral mRNAs escaped decay and robustly translated(70,71). These results suggest that RNA signaling and decay pathways activated by RNase L are complex and the dyamics may vary based on specific activation of RNase L by 2-5A compared to indirect activation by polyI:C as well as cell type differences and abundance of dsRNA-binding proteins including OAS isoforms.We characterized the biochemical nature of avSG formed during RNase L activation using 2-5A, RNase L-cleaved RNAs and SeV infection by adapting a recently published SG purification method and determined interaction among proteins recruited to avSG.Our studies avoided overexpression of G3BP1 which forms SG independent of stimulus by testing interaction with endogenous G3BP1(52).…”

Activation of the antiviral factor RNase L triggers translation of non-coding mRNA sequences

“…Since RNase L dramatically reshapes the transcriptome by degrading mRNA (Burke et al, 2019;Rath et al, 2019) and may itself affect the translation process, we investigated the distribution of ribosomes in cells where RNase L was active by performing ribosome profiling (Ribo-seq) (Ingolia et al, 2009) ( Figure 1A). Active RNase L was shown to cleave both 18S and 28S rRNAs at precise locations (Cooper et al, 2014;Rath et al, 2019;Wreschner et al, 1981). Activation of RNase L is therefore traditionally assessed by using an rRNA cleavage assay (Wreschner et al, 1981).…”

“…This activation can be specifically achieved by transfection with purified 2-5A (prepared as described in Methods) or the double stranded RNA mimic, poly I:C, that also activates additional dsRNA sensors, such as PKR, RIG-I and MDA5 (Chitrakar et al, 2019;de Haro et al, 1996;Martinand et al, 1998). We performed most experiments in the A549 lung carcinoma cell line since these cells had demonstrated robust RNase L activation in previous studies (Burke et al, 2019;Chitrakar et al, 2019;Rath et al, 2019). We found that 4.5 hours treatment of wild type (WT) A549 cells with 1-10 µM 2-5A was sufficient to generate readily detectable rRNA cleavage products by using electrophoretic analysis (BioAnalyzer) of purified total RNA ( Figure 1B).…”

“…RNase L cleaves a broad spectrum of host RNAs, including rRNAs, tRNAs, mRNAs and Y-RNAs (Burke et al, 2019;Donovan et al, 2017;Rath et al, 2015;Rath et al, 2019).…”

“…While cleavage of rRNA was shown to not inhibit the activity of the ribosome (Rath et al, 2019), two recent studies established that, on average, mRNA levels in the cell can be reduced up to ~90% due to RNase L (Burke et al, 2019;Rath et al, 2019). This global effect was termed 2-5A Mediated Decay (2-5AMD), analogous to Nonsense Mediated Decay (NMD) (Rath et al, 2019).…”