N1-methyl-pseudouridine in mRNA enhances translation through eIF2α-dependent and independent mechanisms by increasing ribosome density.
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.
Department of Biochemistry, McGill University, Montréal, Québec H3A 1A3, Canada.|Rosalind and Morris Goodman Cancer Research Centre, Montréal, Québec H3A 1A3, Canada.
Yuri V
YV
Svitkin
Moderna Therapeutics, Cambridge, MA 02139, USA.
Yi Min
YM
Cheng
Moderna Therapeutics, Cambridge, MA 02139, USA.
Tirtha
T
Chakraborty
Moderna Therapeutics, Cambridge, MA 02139, USA.
Vladimir
V
Presnyak
Moderna Therapeutics, Cambridge, MA 02139, USA.
Matthias
M
John
Department of Biochemistry, McGill University, Montréal, Québec H3A 1A3, Canada.|Rosalind and Morris Goodman Cancer Research Centre, Montréal, Québec H3A 1A3, Canada.
Nahum
N
Sonenberg
D000818:Animals; D002460:Cell Line; D002474:Cell-Free System; D004789:Enzyme Activation; D015852:Eukaryotic Initiation Factor-2 / Q000502:physiology*; D005347:Fibroblasts; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D010766:Phosphorylation; D011132:Polyribosomes / Q000378:metabolism*; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D011560:Pseudouridine / Q000031:analogs & derivatives* / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D020871:RNA Stability; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics*; D014162:Transfection; D019892:eIF-2 Kinase / Q000378:metabolism
D015852:Eukaryotic Initiation Factor-2; D012333:RNA, Messenger; C039919:RNA, recombinant; C013608:1-methylpseudouridine; D011560:Pseudouridine; D012313:RNA; D019892:eIF-2 Kinase