Nucleoside modifications in RNA limit activation of 2'-5'-oligoadenylate synthetase and increase resistance to cleavage by RNase L

Abstract

The interferon-induced enzymes 2'-5'-oligoadenylate synthetase (OAS) and RNase L are key components of innate immunity involved in sensory and effector functions following viral infections. Upon binding target RNA, OAS is activated to produce 2'-5'-linked oligoadenylates (2-5A) that activate RNase L, which then cleaves single-stranded self and non-self RNA. Modified nucleosides that are present in cellular transcripts have been shown to suppress activation of several RNA sensors. Here, we demonstrate that in vitro transcribed, unmodified RNA activates OAS, induces RNase L-mediated ribosomal RNA (rRNA) cleavage and is rapidly cleaved by RNase L. In contrast, RNA containing modified nucleosides activates OAS less efficiently and induces limited rRNA cleavage. Nucleoside modifications also make RNA resistant to cleavage by RNase L. Examining translation in RNase L(-/-) cells and mice confirmed that RNase L activity reduces translation of unmodified mRNA, which is not observed with modified mRNA. Additionally, mRNA containing the nucleoside modification pseudouridine is translated longer and has an extended half-life. The observation that modified nucleosides in RNA reduce 2-5A pathway activation joins OAS and RNase L to the list of RNA sensors and effectors whose functions are limited when RNA is modified, confirming the role of nucleoside modifications in suppressing immune recognition of RNA.

Figure 1.

OAS activation by RNA containing modified nucleosides. Purified human OAS1 p42 was activated with RNAs containing either unmodified (U) or the indicated nucleoside modifications and the functionally active 2-5A produced was quantified using a FRET-based assay as described in ‘Materials and Methods’ section. Data shown are mean of three replicates.

Figure 2.

Induction of rRNA cleavage by in vitro transcribed RNA. Unmodified (U) or Ψ-containing RNA encoding firefly luciferase was complexed to lipofectin and delivered to WT or RNase L^−/− MEF cells, as was a no RNA (−) control. At 3 h following transfection, total RNA was recovered from cells. RNA aliquots were separated in an agarose gel and visualized by UV fluorescence. Arrowheads indicate RNase L-specific rRNA cleavage products. Bar graph above bands shows densitometric measurement ± SD of the ratio of cleavage products to uncleaved 18S rRNA, normalized to cells not treated with RNA. Representative data from one of three independent experiments is shown.

Figure 3.

Cleavage of Ψ-containing RNA by RNase L. Purified RNase L was activated on ice by trimer 2-5A prior to mixing with RNA substrates. (A) Cleavage of oligo RNAs [³²P]pC₁₁U₂C₇ (UU) or [³²P]pC₁₁Ψ₂C₇ (ΨΨ) by RNase L. Reactions were stopped at the indicated time by addition of loading buffer, and reactions were separated by PAGE and visualized by phosphor-storage radiography. Representative data from one of three independent experiments is shown. (B) Quantification of band intensities. Values were normalized to the values obtained in the 30 min reaction not containing RNase L. Data represents average ± SEM of n = 3 experiments. Asterisks indicate P < 0.05. (C) Cleavage of metabolically [³²P]-labeled unmodified (U) or Ψ-containing firefly luciferase mRNA by RNase L. Reactions were stopped at indicated times. Aliquots of isolated RNA from each reaction were separated by PAGE and visualized by phosphor-storage radiography. Representative data from one of three independent experiments is shown.

Figure 4.

Stability of Ψ-containing RNA. Unmodified (U) or Ψ-containing RNAs encoding firefly or Renilla luciferases were mixed 1:1 and either added to RRL (A) or nucleofected to HEK293T cells (B). At the indicated time points, RNA was recovered and detected by northern blotting. Radiolabeled DNA probes corresponding to the coding sequence of firefly and Renilla luciferases were mixed prior to northern hybridization (A) or used separately to probe duplicate northern blots from aliquots of the recovered RNA (B). Bar graphs above the images show densitometric measurement of the uncleaved firefly luciferase mRNA. Data shown is representative of at least five independent experiments. (C) Unmodified (U) or Ψ-containing in vitro transcribed RNA encoding firefly luciferase was delivered to WT or RNase L^−/− MEF cells by nucleofection. Cells were lysed at 0.2, 1, 3, 6 or 24 h following transfection, total RNA was recovered, and luciferase RNA was assessed by northern blotting. Radiolabeled DNA corresponding to the firefly luciferase coding sequence was used as probe. Bar graph above bands shows densitometric measurement of the uncleaved RNA. Representative data is shown from one of three independent experiments.

Figure 5.

Translation of unmodified and Ψ-containing mRNA in WT and RNase L^−/− cells and mice. Unmodified (U) or Ψ-containing in vitro transcribed mRNA encoding firefly luciferase was complexed to lipofectin and delivered to WT and RNase L^−/− MEF cells or mice. Luciferase activity was measured in aliquots of cell or spleen lysate. (A) MEF cells lysed 5 h following transfection. Values presented are luciferase relative light units (RLU) in 2 µl of the total 20 µl cell lysate. Error bars indicate SEM of quadruplicate wells from one representative of at least six independent experiments. (B) Lipofectin-complexed mRNA was delivered by tail vein injection into mice. Mice were sacrificed at 4 h post-transfection and their spleens were homogenized in lysis buffer. Values presented are luciferase RLU in 1/5 of the total 200 µl spleen lysate. Error bars represent SEM of n = 3 mice.

Figure 6.

Translation of Ψ-containing mRNA in cell culture. Unmodified (U) and Ψ-containing mRNA were complexed to lipofectin and delivered to HEK293T cells. (A) Renilla luciferase activity was assessed in aliquots of cell lysate. Data displayed is mean ± SEM from four replicates, each performed in duplicate. (B) Cells were pulsed for the last 3 h with ³⁵S-methionine/cysteine prior to lysis at the indicated time points. Renilla luciferase protein was immunoprecipitated from cell lysates, separated by PAGE and then visualized by fluorography. Data shown is one of four replicates and is representative of three independent experiments.

Figure 7.

Structures and base pairing of uridine and pseudouridine. In pseudouridine, uracil is linked to ribose via C5 instead of the N1 linkage found in uridine (C5 and N1 are indicated in bold type). Hydrogen bonds between adenosine and uridine or pseudouridine are indicated by dotted lines. Additional hydrogen bonding potential of pseudouridine is indicated by dashed arrow.