Abstract
mRNAs lacking 5′ untranslated regions (leaderless mRNAs) are molecular relics of an ancient translation initiation pathway. Nevertheless, they still represent a significant portion of transcriptome in some taxons, including a number of eukaryotic species. In bacteria and archaea, the leaderless mRNAs can bind non-dissociated 70 S ribosomes and initiate translation without protein initiation factors involved. Here we use the Fleeting mRNA Transfection technique (FLERT) to show that translation of a leaderless reporter mRNA is resistant to conditions when eIF2 and eIF4F, two key eukaryotic translation initiation factors, are inactivated in mammalian cells. We report an unconventional translation initiation pathway utilized by the leaderless mRNA in vitro, in addition to the previously described 80S-, eIF2-, or eIF2D-mediated modes. This mechanism is a bacterial-like eIF5B/IF2-assisted initiation that has only been reported for hepatitis C virus-like internal ribosome entry sites (IRESs). Therefore, the leaderless mRNA is able to take any of four different translation initiation pathways in eukaryotes.
Highlights
Translational properties of both prokaryotic and eukaryotic mRNAs are largely dictated by their 5′untranslated regions (5′UTRs)
We show that cI-derived leaderless transcript encoding firefly luciferase can direct translation in living mammalian cells under conditions of severe stress when the canonical cap-dependent ribosomal scanning is strongly compromised
To perform such an analysis, we prepared a firefly luciferase (Fluc) reporter construct starting with the cI mRNA 5′terminal sequence (40 nt in length, Supplementary Fig. 1)
Summary
Translational properties of both prokaryotic and eukaryotic mRNAs are largely dictated by their 5′untranslated regions (5′UTRs). Nuclear-encoded leaderless transcripts are widely represented across a number of primitive unicellular organisms[4,5] This peculiar class of mRNAs is present in all three domains of life. The ability of the lambda phage leaderless cI mRNA to bind directly to non-dissociated 70 S ribosomes in the presence of fMet-tRNAMetf was initially reported for bacterial systems[10]. This binding did not require any additional protein factors. Translational properties of the leaderless mRNA have never been studied in living cells of a higher eukaryote
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