TY - JOUR
T1 - Incorporation of pseudouridine into mRNA enhances translation by diminishing PKR activation
AU - Anderson, Bart R.
AU - Muramatsu, Hiromi
AU - Nallagatla, Subba R.
AU - Bevilacqua, Philip C.
AU - Sansing, Lauren H.
AU - Weissman, Drew
AU - Karikó, Katalin
N1 - Funding Information:
The National Institutes of Health (R01AI50484, R21DE019059 to D.W., T32GM07229, T32DK07748, T32RR007063 to B.R.A., R42HL87688 to K.K. and R01GM058709 to P.C.B.). Funding for open access charge: National Institutes of Health grant R42HL87688.
PY - 2010/5/10
Y1 - 2010/5/10
N2 - 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.
AB - 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.
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U2 - 10.1093/nar/gkq347
DO - 10.1093/nar/gkq347
M3 - Article
C2 - 20457754
AN - SCOPUS:77957242130
SN - 0305-1048
VL - 38
SP - 5884
EP - 5892
JO - Nucleic acids research
JF - Nucleic acids research
IS - 17
M1 - gkq347
ER -