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-2 alpha), 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.
