Nucleoside modifications modulate activation of the protein kinase PKR in an RNA structure-specific manner

Abstract

The human interferon-induced protein kinase PKR is a key component of innate immunity, a process in which it senses pathogenic RNA. PKR consists of an N-terminal dsRNA-binding domain (dsRBD) and a C-terminal kinase domain. Upon binding long (>33 base pairs) stretches of pathogenic dsRNA, PKR undergoes autophosphorylation, which activates it to phosphorylate eIF2alpha, leading to inhibition of translation initiation. Many cellular and viral transcripts contain nucleoside modifications, and these could affect PKR activation. For example, a 5'-triphosphate confers the ability of relatively unstructured transcripts to activate PKR. Effects of internal RNA modifications on PKR activation have not been reported. Herein, PKR activation by ssRNA and dsRNA containing internal nucleobase, sugar, and phosphodiester modifications is analyzed. We find that for 5'-triphosphate-containing ssRNA, most base and sugar modifications abrogate activation, although 2'-fluoro-modified ssRNA does not, indicative of a critical role for hydrogen bonding at the ribose sugar. In the case of dsRNA, a more limited set of nucleoside modifications affect PKR activation. Watson-Crick base-pairing is required for activation, and some minor groove modifications abrogate activation while major groove modifications have little effect. Surprisingly, GU wobble pairs also largely abrogate dsRNA-mediated activation when present at modest levels. Modifications to dsRNA that abrogate activation have no significant effect on dsRBD binding, allowing such RNAs to act as inhibitors and suggesting a nonequivalence of binding ability and activation. Overall, the findings indicate that nucleoside modifications and wobble pairing may serve to discriminate self-RNA and pathogenic RNA in innate immunity.

FIGURE 1.

Effect of ssRNA-47 modifications on PKR activation. (A) Schematic of PKR protein. PKR is 551 residues and has two tandem N-terminal dsRNA-binding motifs (dsRBM1 and dsRBM2) that comprise its dsRNA-binding domain (dsRBD) and multiple C-terminal kinase subdomains that confer catalytic activity. P20 is the protein that goes from 1 to 184, and so contains the dsRBD. (B) Secondary structural model of ssRNA-47. This structure was derived from structure-mapping experiments in conjunction with energy minimization from mfold v3.2 as described in Nallagatla et al. (2007). (C) Modified nucleosides studied. (D) Activation assays for modified ssRNAs (10% SDS-PAGE). RNA concentrations are 0.16, 0.31, 0.63, 1.25, 2.5, and 5 μM. A no-RNA-added lane is provided. Phosphorylation activities are presented below each gel lane. Phosphorylation activities were normalized to 0.1 μM dsRNA-79 and rounded off to the nearest integer. Unmodified ssRNA-47 was included as a control in each separate experiment.

FIGURE 2.

Effect of dsRNA-47 modifications on PKR activation. (A) Sequence of dsRNA-47. Only the top strand was modified. (B,C) Activation assays for modified dsRNA-47 (10% SDS-PAGE) (B) and s2U-modified dsRNA-79 (C). For both panels, the radiolabeled top strand was modified and annealed with a slight excess of unmodified and unlabeled CIP-treated bottom strand (see Materials and Methods). The dsRNA concentrations are 0.016, 0.032, 0.062, 0.125, 0.25, and 0.5 μM. A no-RNA-added lane is provided. Phosphorylation activities are normalized to 0.1 μM dsRNA-79 and rounded off to the nearest integer. Unmodified dsRNA-47 was included as a control in each separate experiment.

FIGURE 3.

Effect of A–s4U, G–s4U, and GU pairs on PKR activation by dsRNA-47. (A) Structures of A–s4U, G–s4U, and GU base pairs. (B) dsRNA-47 variants having 12 s4U modifications in the top strand and variable number of opposing A or G bases in the bottom strand. The A–s4U pairs are boxed, and the G–s4U pairs are shaded with the G in the GU wobbles shown by lower-case letters. The s4U-modified dsRNA1 duplex, “dsRNA1–s4U,” has a bottom strand with all As opposite the 12 s4U substitutions. (The unmodified version of dsRNA1 is the same sequence used in Fig. 2.) dsRNA2–s4U is a sequence variant in which the bottom strand has two As and 10 Gs opposite the 12 s4U substitutions, while dsRNA3–s4U is a sequence variant in which the bottom strand has five As and seven Gs opposite the 12 s4U substitutions. The percent modification or GU wobble pairing is provided in panels C and D. (C) PKR activation assays by s4U-modified dsRNAs (10% SDS-PAGE). s4U-modified versions of dsRNA1, dsRNA2, and dsRNA3 as well as an unmodified dsRNA1 were tested for PKR activation. The dsRNA concentrations are 0.016, 0.032, 0.062, 0.125, 0.25, and 0.5 μM. (D) PKR activation assay by dsRNA1 in which top strand has no modification and bottom strand has no A-to-G changes, and dsRNA2 in which top strand has no modifications and bottom strand has 10 A-to-G changes. The dsRNA concentrations are 0.032, 0.062, 0.125, 0.25, and 0.5 μM. A no-RNA-added lane is provided. Phosphorylation activities are normalized to 0.1 μM dsRNA-79 and rounded off to the nearest integer.

FIGURE 4.

Effect of different levels of A–s2U base-pairing on PKR activation by dsRNA-47. (A) Structures of A–U and A–s2U base pairs. (B) dsRNA-47 variants having variable numbers of s2U modifications in the top strand. dsRNA1–s2U has 12 s2U substitutions (26% modification) in the top strand. (The unmodified version of dsRNA1 is the same sequence used in Fig. 2.) dsRNA4–s2U is a sequence variant in which the top strand has six s2U substitutions (13% modification), while dsRNA5–s2U has 3 s2U substitutions (6% modification) in the top strand. (C) PKR activation assays by these RNAs (10% SDS-PAGE). Unmodified and s2U-modified versions of dsRNA1, dsRNA4, and dsRNA5 were tested for PKR activation. The dsRNA concentrations are 0.016, 0.032, 0.062, 0.125, 0.25, and 0.5 μM. A no-RNA-added lane is provided. Phosphorylation activities are normalized to 0.1 μM dsRNA-79 and rounded off to the nearest integer.

FIGURE 5.

Binding of unmodified and modified dsRNA-47 to P20 by mobility-shift assays. Trace amounts of radiolabeled modified dsRNAs were mixed with P20 and analyzed by 10% native PAGE (79:1 crosslink). Modified (mod.) top strand (p*TS) oligonucleotide was 5′-³²P-labeled and pre-annealed to excess unlabeled and unmodified (unmod.) bottom strand (BS) oligonucleotide. Formation of duplex was confirmed by a microshift of the top strand upon addition of bottom strand. (Compare first and second lanes in each RNA set.) PS-U/PS-A represents dsRNA-47 in which top strand has PS-U modification and bottom strand has PS-A modification. Protein concentrations used are 0.005, 0.01, 0.02, 0.04, 0.08, 0.16, 0.32, 0.63, 1.25, 2.5, and 5 μM.

FIGURE 6.

Inhibition of PKR activation by modified ssRNA-47 and dsRNA-47. (A) Inhibition of PKR activation by modified ssRNA-47s. To each lane containing 5 μM of unmodified activating ssRNA-47, an equal concentration of modified ssRNA was added, and PKR activation was assayed (10% SDS-PAGE). Phosphorylation activities are normalized to 0.1 μM dsRNA-79 and rounded off to the nearest integer. (B) Inhibition of PKR activation by modified dsRNA-47s. To each lane containing 0.125 μM of unmodified activating dsRNA-47, 0.125 or 0.5 μM modified dsRNA was added, and PKR activation was assayed (10% SDS-PAGE). Phosphorylation activities are normalized to 0.1 μM dsRNA-79 and rounded off to the nearest integer.