Four translation initiation pathways employed by the leaderless mRNA in eukaryotes

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.

A Retrospective on eIF2A—and Not the Alpha Subunit of eIF2

“…Phosphorylation of the α subunit of the eIF2 complex inhibits eIF2B-mediated exchange of eIF2•GDP to eIF2•GTP [66,72,73], thus reducing the formation of the active TC [63,64]. However, translation of a subset of cellular and viral mRNAs appeared to be resistant to eIF2α phosphorylation, despite requiring Met-tRNA i [3,[48][49][50][51][52][53][54][55][56][57][58]. These mRNAs in many instances do not require m 7 G cap recognition by eIF4E, and their translation may rely on alternative initiation mechanisms such as internal initiation and/or re-initiation [3,[50][51][52][53].…”

“…However, translation of a subset of cellular and viral mRNAs appeared to be resistant to eIF2α phosphorylation, despite requiring Met-tRNA i [3,[48][49][50][51][52][53][54][55][56][57][58]. These mRNAs in many instances do not require m 7 G cap recognition by eIF4E, and their translation may rely on alternative initiation mechanisms such as internal initiation and/or re-initiation [3,[50][51][52][53]. It was found that a subset of factors, i.e., Ligatin/eIF2D [59,60], the oncogene MCT-1 and DENR (together) [59] as well as eIF5B (alone) [49,74,75] can promote efficient recruitment of Met-tRNA i to 40S/mRNA complexes under conditions of inhibition of eIF2 activity, or its absence.…”

“…With the discovery of eIF2, the research on eIF2A languished. However, further discovery of a variety of alternative pathways for eukaryotic initiation (that may not rely on eIF2 and/or the presence of the m 7 G cap structure at the 5 end of eukaryotic mRNAs) [3,[48][49][50][51][52][53][54][55][56][57][58]] generated a renewed interest in eIF2A and the role it might play in these alternative initiation events. In addition, it appeared that several other factors, and, specifically, Ligatin/eIF2D [59,60] and the complex of the oncogene product, MCT-1, and density regulated protein (DENR) [59] can promote recruitment of Met-tRNA i to some 40S/mRNA complexes under conditions of inhibition of eIF2 activity.…”

Functional Cyclization of Eukaryotic mRNAs

“…Interestingly, translation of an mRNA with a very short leader (5 nt) revealed the weakest acceleration effect. This suggests, in the context of the cyclic reinitiation model, that the distance between the cyclization point (cap) and AUG can be too small for CLAR to occur efficiently while still being enough for the efficient de novo cap-dependent initiation (Figure 4b, see also [51]).…”

Computational design and interpretation of single-RNA translation experiments

“…15), of the simulated fluorescence intensities, and from G(τ ) we estimate the time 250 lag, τ F CS , at which correlations disappear (see Fig 1C and Methods). In the ROA 251 approach, we simulate the addition of a chemical compound, such as Harringtonine, 252 which binds the 60S ribosome subunit and prevents ribosome assembly [26], and we 253 record the average time, τ ROA , at which protein fluorescence disappears from the RNA 254 (see Fig 1D and Methods). To approximate variability in the specific time at which the 255 drug reaches the mRNA and blocks ribosome initiation, we assume that the time of 256 initiation blockage occurs at a normally distributed time of 60 ± 10 seconds [27].…”