Incorporation of pseudouridine into mRNA enhances translation by diminishing PKR activation

TLDR

Previous studies have shown that replacing uridines with pseudouridines in in‑vitro transcribed mRNA increases translation, but the underlying mechanism was unknown. The study aims to determine whether pseudouridine incorporation reduces PKR activation and thereby enhances mRNA translation. The authors validated PKR’s role by demonstrating that pseudouridine‑ and uridine‑containing RNAs are translated equally in PKR‑knockout cells. They found that uridine‑containing mRNAs strongly activate PKR, trigger eIF‑2α phosphorylation and translational repression, whereas pseudouridine diminishes PKR activation, preventing repression, and that this effect is lost in PKR‑knockout cells, confirming PKR mediates the enhanced translation.

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.