Dynamic refolding of IFN-γ mRNA enables it to function as PKR activator and translation template

Abstract

Interferon-gamma mRNA activates the RNA-dependent protein kinase PKR, which in turn strongly attenuates translation of interferon-gamma mRNA. Unlike riboswitches restricted to noncoding regions, the interferon-gamma RNA domain that activates PKR comprises the 5' UTR and 26 translated codons. Extensive interferon-gamma coding sequence is thus dedicated to activating PKR and blocking interferon-gamma synthesis. This implies that the PKR activator is disrupted by ribosomes during translation initiation and must refold promptly to restore PKR activation. The activator structure harbors an essential kink-turn, probably to allow formation of a pseudoknot that is critical for PKR activation. Three indispensable short helices, bordered by orientation-sensitive base pairs, align with the pseudoknot stem, generating RNA helix of sufficient length to activate PKR. Through gain-of-function mutations, we show that the RNA activator can adopt alternative conformations that activate PKR. This flexibility promotes efficient refolding of interferon-gamma mRNA, which is necessary for its dual function as translation template and activator of PKR, and which thus prevents overexpression of this inflammatory cytokine.

IL-1-induced Post-transcriptional Mechanisms Target Overlapping Translational Silencing and Destabilizing Elements in IκBζ mRNA*

“…Activation of translation of negative regulators by IL-1 (Table 1) may achieve the same goal, eventual termination of the response, as IFN-␥-induced inhibition of translation; IFN-␥, which itself is translationally controlled by a PKR-activating sequence spanning part of its 5Ј UTR and coding sequences (72,73), restricts translation of some of its target genes through a heteromeric complex assembled at the target mRNAs (74).…”

Activation of PKR by RNA misfolding: HDV ribozyme dimers activate PKR

“…These three helical segments together would make a 56-bp dsRNA, which would be a strong PKR activator, as observed herein. The ability of multiple shorter helical fragments to promote activation has been noted previously in aptamers, pseudoknots, TAR, and the HCV IRES (Bevilacqua et al 1998;Cohen-Chalamish et al 2009;Shimoike et al 2009;Heinicke 2010;Toroney et al 2010). We note that similar activation of PKR by dimers of 1/99 D3 bp, WT, and +3 bp constructs at 0.1 mM RNA dimer (Supplemental Fig.…”

“…In addition, PKR is known to be activated by highly structured RNA domains of hepatitis C virus (HCV) (Shimoike et al 2009;Toroney et al 2010) and a pseudoknotted human IFN-g-mRNA (Ben-Asouli et al 2002;Cohen-Chalamish et al 2009). Unlike the above activators, the HDV ribozyme is semiglobular, although it contains a protruding 13-bp pairing region (P4).…”

Allele-specific SHAPE-MaP assessment of the effects of somatic variation and protein binding on mRNA structure

“…TPT1 mRNA activates the double-stranded RNA (dsRNA) recognition protein PKR (Bommer et al 2002;Nussbaum et al 2002). PKR primarily Cold Spring Harbor Laboratory Press on May 12, 2018 -Published by rnajournal.cshlp.org Downloaded from recognizes viral dsRNA and inhibits viral translation, but PKR also interacts with a variety of cellular dsRNA-containing mRNAs, including the TNF-α and IFN-γ (Osman et al 1999;Cohen-Chalamish et al 2009;Hull and Bevilacqua 2016). Activation of PKR depends on a dsRNA region of at least 30 bases to allow binding of two or more PKR molecules (Manche et al 1992;Zheng and Bevilacqua 2004;Lemaire et al 2008).…”