Incorporation of pseudouridine into mRNA enhances translation by diminishing PKR activation

By Bart R. Anderson, Hiromi Muramatsu, S. Nallagatla, P. Bevilacqua, Lauren H. Sansing, D. Weissman, K. Karikó. Nucleic Acids Research, 2010

616 citations recorded by the source corpus.

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.

Subfields: Molecular Biology

View Paper: https://www.semanticscholar.org/paper/43378705d3b663c881e981da3ac58da8af80cca3

How researchers see this paper

The researchers whose trusted networks rank it highest, strongest advocate first. Every researcher gets a different order.

In Molecular Biology

1. Michael J. Hogan
2. N. Pardi
3. K. Karikó
4. Ugur Sahin
5. Ö. Türeci
6. D. Weissman
7. Hiromi Muramatsu
8. G. B. Robb

Citations in the corpus

• mRNA Vaccines in the COVID-19 Pandemic and Beyond. (2021, background): “, protein kinase RNA-activated (PKR) and 2′-5′-oligoadenylate synthetase (OAS)] (61, 62), leading to a signaling cascade that significantly dampens protein expression (3).”
• mRNA capping: biological functions and applications (2016, positive): “The use of m5CTP and pseudouridine triphosphate instead of CTP and UTP, respectively, in transcription has been shown to reduce the TLR-induced immune response and inhibition of protein synthesis (161– 163).”
• mRNA-based therapeutics — developing a new class of drugs (2014, positive): “75 ) , and some of them, including pseudouridine and 2-thiouridine, were shown to make IVT mRNA undetectable by RIG-I and PKR 76–78 .”
• Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA (2011, methodology): “We previously demonstrated that incorporation of m5C and into long mRNA reduced its ability to activate PKR (6).”