Incorporation of pseudouridine into mRNA enhances translation by diminishing PKR activation

Abstract

Previous studies have shown that the translation level of in vitro transcribed messenger RNA (mRNA) is enhanced when its uridines are replaced with pseudouridines; however, the reason for this enhancement has not been identified. Here, we demonstrate that in vitro transcripts containing uridine activate RNA-dependent protein kinase (PKR), which then phosphorylates translation initiation factor 2-alpha (eIF-2α), and inhibits translation. In contrast, in vitro transcribed mRNAs containing pseudouridine activate PKR to a lesser degree, and translation of pseudouridine-containing mRNAs is not repressed. RNA pull-down assays demonstrate that mRNA containing uridine is bound by PKR more efficiently than mRNA with pseudouridine. Finally, the role of PKR is validated by showing that pseudouridine- and uridine-containing RNAs were translated equally in PKR knockout cells. These results indicate that the enhanced translation of mRNAs containing pseudouridine, compared to those containing uridine, is mediated by decreased activation of PKR.

Intratumoral Delivery of TriMix mRNA Results in T-cell Activation by Cross-Presenting Dendritic Cells

“…negatively affects its translation, implying that stronger TiDC activation could be obtained using "modified" mRNA that does not trigger PRRs (40)(41)(42). If PRRs are triggered, the effects observed in this study may be due to the delivery of mRNA rather than the delivery of TriMix mRNA.…”

mRNA-engineered mesenchymal stem cells for targeted delivery of interleukin-10 to sites of inflammation

“…For instance, a recent study showed that incorporation of C improved protein expression efficiency by reducing activation of RNA-activated protein kinase (PKR). 37 Warren et al reported that complete substitution of cytidine with 5mC in C-containing in vitro-transcribed RNA can further increase expression efficiency. 35 Hence, we tested the impact of substituting uridine for C, cytidine for 5mC, or both on protein expression efficiency in mRNA-transfected MSCs (supplemental Figure 1D-E).…”

“…As shown in Figure 4, the administered native MSCs were observed in the BM of both healthy (nonirradiated) and g-irradiated mice (68. 37 Figure 4B). These data strongly suggest that equipping MSCs with the homing ligands PSGL-1/SLeX via mRNA transfection significantly improves their homing to the BM, especially under inflammatory conditions.…”

Post-transcriptional modifications in development and stem cells

“…4) (Schwartz et al, 2014a). Other studies reported an impact on protein translation, where the presence of Ψ in CDS of mRNA increased translation efficiency (Anderson et al, 2010;Kariko et al, 2008). Mass spectrometry sequencing as well as genetic ablation experiments in yeast showed that Ψ in stop codons decreases the efficiency of translation termination (Fig.…”