Interaction of PKR with single-stranded RNA

Abstract

Although the antiviral kinase PKR was originally characterized as a double-stranded RNA activated enzyme it can be stimulated by RNAs containing limited secondary structure. Single-stranded regions in such RNAs contribute to binding and activation but the mechanism is not understood. Here, we demonstrate that single-stranded RNAs bind to PKR with micromolar dissociation constants and can induce activation. Addition of a 5′-triphosphate slightly enhances binding affinity. Single-stranded RNAs also activate PKR constructs lacking the double-stranded RNA binding domain and bind to a basic region adjacent to the N-terminus of the kinase. However, the isolated kinase is not activated by and does not bind single-stranded RNA. Photocrosslinking measurements demonstrate that that the basic region interacts with RNA in the context of full length PKR. We propose that bivalent interactions with the double stranded RNA binding domain and the basic region underlie the ability of RNAs containing limited structure to activate PKR by enhancing binding affinity and thereby increasing the population of productive complexes containing two PKRs bound to a single RNA.

The search for a PKR code—differential regulation of protein kinase R activity by diverse RNA and protein regulators

“…Single-stranded RNAs. Long ssRNAs lacking any discernable secondary structures (such as homopolymeric U30) have also been recently shown to activate PKR in vitro (Toroney et al 2012;Mayo and Cole 2017). Dimerization of PKR on this ssRNA was observed, corroborated by weak PKR autophosphorylation.…”

“…This led to models where RNA dimerization or end-to-end coaxial stacking dictate PKR activation, by presenting a near A-form extended duplex structure long enough to bind two PKR monomers (Heinicke et al 2009;Nallagatla et al 2011). Recently, short dsRNA flanked by singlestranded termini, termed ss-dsRNA, have been identified as a new class of PKR activators (Nallagatla et al 2007;Mayo and Cole 2017). Below we provide specific examples of these divergent RNA activators and discuss their structural features related to PKR activation.…”

“…Initially uncovered from in vitro selection experiments that selected for dsRBM/PKR-binding RNAs, RNAs that contain both dsRNA and ssRNA elements have emerged as new, robust PKR activators (Zheng and Bevilacqua 2004;Nallagatla et al 2007). An imperfect 16bp or a perfect 15-bp duplex stem flanked by single stranded tails (ss-dsRNA) of 15 nt length on both 5 ′ and 3 ′ ends strongly bind and activate PKR autophosphorylation (Nallagatla et al 2007;Toroney et al 2012;Mayo et al 2016;Mayo and Cole 2017). Interestingly, a minimal 10bp stem is necessary for activation, and the length but not the sequence of the flanking ssRNA is critical for PKR activation (Mayo et al 2016).…”

“…Photochemical cross-linking experiments with 4-thio-U labeled ss-dsRNA identified direct contacts with the dsRBMs as well as the basic patch in the linker that immediately precedes the KD. This led to an intriguing model where a bivalent interaction of ss-dsRNA with both the dsRBMs (via the stem) and the KD (via the flanking ends) drive activation (Mayo and Cole 2017).…”

“…Importantly, ssRNAs bound and activated the isolated KD, suggesting that activation is independent of dsRBM binding. Instead, the basic patch neighboring the KD was found to be strictly required for efficient recognition and autophosphorylation of PKR (Mayo and Cole 2017). Similarly to ss-dsRNAs, uncertainty remains whether a 5 ′ -ppp is needed for proper activation.…”