Multiple innate defense pathways exist to recognize and defend against foreign nucleic acids. Unlike innate immune receptors that recognize structures specific for pathogens that are not shared by mammalian hosts — for example, toll-like receptor (TLR)4-lipopolysaccharide, TLR5-flagellin, NOD1 and 2-peptidoglycan — all nucleic acids are made from four components that are identical from bacteria to man. Nucleoside modifications are prevalent in nature but vary greatly in their distribution and frequency, and therefore could serve as patterns for recognition of pathogenic nucleic acids. The presence of modified nucleosides in RNA reduces the activation of RNA-sensing TLRs and retinoic acid inducible gene I (RIG-I), which initiate signaling cascades following activation and result in transcription of pro-inflammatory genes. Unexpectedly, translation of in vitro transcribed mRNA is enhanced by incorporation of modified nucleosides, but the mechanism responsible for this enhanced translation has not been identified. To identify the pathways responsible for enhanced translation of modified nucleoside-containing mRNA, we studied two cytoplasmic RNA-sensing innate defense mechanisms known to influence translation, the RNA-dependent protein kinase (PKR) pathway and the 2-5A system (oligoadenylate synthetase [OAS] and RNase L). Using purified protein in vitro, cell culture, and in vivo mouse studies, we show that unmodified in vitro transcribed mRNA activates PKR and OAS and is rapidly cleaved by RNase L. However, we show that incorporation of modified nucleosides into in vitro transcribed mRNA reduces each of these pathways. Furthermore, we demonstrate that these pathways are necessary for enhanced translation of mRNA containing modified nucleosides. Additionally, we demonstrate that the presence of pseudouridine in in vitro transcripts increases mRNA half-life following delivery. From these data, we conclude that unmodified in vitro transcribed mRNA is stimulatory to the cytoplasmic RNA sensors PKR and OAS. This stimulation is reduced by the presence of modified nucleosides. The enhanced translation of mRNA containing modified nucleosides results from reduced PKR and OAS activation. These data support a larger interpretation that the absence or reduction in frequency of modified nucleosides in RNA is a common pattern for recognition of pathogenic RNA by numerous innate defense systems
