Incorporation of pseudouridine into mRNA enhances translation by diminishing PKR activation

Anderson, Bart R.
Muramatsu, Hiromi
Nallagatla, Subba R.
Bevilacqua, Philip C.
Sansing, Lauren H.
Weissman, Drew
Karikó, Katalin

¹Department of Medicine,
²Department of Neurosurgery, University of Pennsylvania, Philadelphia, PA,
³Department of Chemistry, Pennsylvania State University, University Park, PA and
⁴Department of Neurology, University of Pennsylvania, Philadelphia, PA, USA

^*To whom correspondence should be addressed. Tel: +1 215 662 7927; Fax: +1 215 349 8157; Email: [email protected]

Received 8 February 2010; revised 8 April 2010; accepted 20 April 2010

Nucleic Acids Research 38(17):p 5884-5892, September 2010. | DOI: 10.1093/nar/gkq347

ABSTRACT

Previous studies have shown that the translation level ofin vitrotranscribed 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 thatin vitrotranscripts containing uridine activate RNA-dependent protein kinase (PKR), which then phosphorylates translation initiation factor 2-alpha (eIF-2α), and inhibits translation. In contrast,in vitrotranscribed 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.