Four translation initiation pathways employed by the leaderless mRNA in eukaryotes

Figure 1. Leaderless mRNA translation is relatively resistant to translation initiation inhibitors in transfected mammalian cells. (a) Schematic representation of the Fleeting mRNA transfection procedure (FLERT). (b) Translation efficiency of the reporter mRNAs constructs. HEK293T cells were transfected with ARCA- (Anti-Reverse Cap Analog) or A-capped polyadenylated Fluc constructs having indicated 5′ leaders, along with the similarly prepared ARCA-capped Actin-Rluc mRNA. In the cIstop-Fluc construct, the 5′ terminal AUG was replaced with UAA. Fluc activity was analyzed after 2 h incubation, normalized to Rluc activity units and represented as a ratio to the value for the capped cI-Fluc mRNA. The mean absolute value for Fluc in this case was 221,805 luciferase units, Rluc – 16,238,226 units, while the background (no-transfection control) values were 60 and 150 units, respectively. Cap-dependence (i.e. ratio of translation efficiency of the ARCA- vs. A-capped mRNAs) is indicated at the bottom. (c) Schematic representation of inhibitors action on the translation initiation pathway. (d) Translation inhibition by the drugs as revealed by the FLERT technique. Luciferase activities are…

Figure 2. Leaderless mRNA translation is relatively resistant to selective 60 S targeting inhibitors. (a) Schematic representation of the inhibitors action. Note that harringtonine and T-2 toxin are unable to enter elongating ribosomes and pre-associated vacant 80 S particles, while puromycin binds the ribosomes at any stage. (b) Translation inhibition by the drugs as revealed by the FLERT technique. Luciferase activities are normalized to those in wells without the inhibitors.

Figure 3. Peculiar properties of the leaderless mRNA translation in the cell-free systems. (a) Selective stimulation of the cI-Fluc mRNA translation by elevated magnesium concentration in cytoplasmic extract of Krebs-2 mouse ascites carcinoma cells. Note that only the concentration of Mg(OAc)2 in the reaction buffer is shown (without considering an endogenous Mg2+ concentration in the extract). The mean absolute values for cI-Fluc and (CAA)5cI-Fluc at 0.8 mM Mg2+ were 40,722 and 255,720 luciferase units, respectively. (b) Differential effects of the small molecule drugs and some translation initiation factors on translation of the cI-Fluc and (CAA)5cI-Fluc mRNAs in Krebs-2 cells extract (p < 0.05). Final concentrations of the additives were as follows: 1.5 mM spermidine-HCl; 250 μM NSC119889; 25 μM salubrinal; 10 ng/μl eIF1; 30 ng/μl eIF5; 50 ng/μl eIF5B. (c) Relative resistance of the leaderless mRNA translation to inhibition by recombinant eIF2αβ/aIF2γ chimeric protein in the yeast cell-free system. The luciferase activity values for the cI-Fluc mRNA were normalized to that of the (CAA)5cI-Fluc mRNA. The mean absolute values for cI-Fluc and (CAA)5cI-Fluc at the control point were 104,678…

Figure 4. The fourth translation initiation pathway for the leaderless mRNA. (a) 48 S pre-initiation complex reconstitution from purified mammalian components on capped cIlacZ transcript. The assembled complexes were visualized by the toe-printing assay. Cross signs denote the components added to the reaction mixture. Sequencing lanes obtained with the same primer and the corresponding cDNA are shown on the left. (b) Schematic representation of four translation initiation pathways employed by the leaderless mRNA in eukaryotes.