N1-methyl-pseudouridine in mRNA enhances translation through eIF2α-dependent and independent mechanisms by increasing ribosome density

• Yuri V. Svitkin ^[1] ; Yi Min Cheng ^[2] ; Tirtha Chakraborty ^[2] ; Vladimir Presnyak ^[2] ; Matthias John ^[2] ; Nahum Sonenberg ^[1]
  1. [1] Department of Biochemistry, McGill University, Montréal, Québec H3A 1A3, Canada; Rosalind and Morris Goodman Cancer Research Centre, Montréal, Québec H3A 1A3, Canada
  2. [2] Moderna Therapeutics, Cambridge, MA 02139, USA
• Localización: Nucleic acids research, ISSN 0305-1048, Vol. 45, Nº. 10, 2017, págs. 6023-6036
• Idioma: inglés
• Enlaces
  • Texto completo
• Resumen

  • Certain chemical modifications confer increased stability and low immunogenicity to in vitro transcribed mRNAs, thereby facilitating expression of therapeutically important proteins. Here, we demonstrate that N1-methyl-pseudouridine (N1mΨ) outperforms several other nucleoside modifications and their combinations in terms of translation capacity. Through extensive analysis of various modified transcripts in cell-free translation systems, we deconvolute the different components of the effect on protein expression independent of mRNA stability mechanisms. We show that in addition to turning off the immune/eIF2α phosphorylation-dependent inhibition of translation, the incorporated N1mΨ nucleotides dramatically alter the dynamics of the translation process by increasing ribosome pausing and density on the mRNA. Our results indicate that the increased ribosome loading of modified mRNAs renders them more permissive for initiation by favoring either ribosome recycling on the same mRNA or de novo ribosome recruitment.