Internal translation initiation and eIF4F/ATP-independent scanning of mRNA by eukaryotic ribosomal particles

Abstract

The recombinant mRNAs with 5'-untranslated region, called omega leader, of tobacco mosaic virus RNA are known to be well translated in eukaryotic cell-free systems, even if deprived of cap structure. Using the method of primer extension inhibition (toe-printing), the ribosomal particles were shown to initiate translation at uncapped omega leader when its 5'-end was blocked by a stable RNA-DNA double helix, thus providing evidence for internal initiation. The scanning of the leader sequence and the formation of ribosomal 48S initiation complexes at the initiation AUG codon occurred in the absence of ATP-dependent initiation factor eIF4F, as well as without ATP. The latter results implied the ability of ribosomal initiation complexes for ATP-independent, diffusional wandering (also called bi-directional movement) along the leader sequence during scanning.

Figure 1. Blocking of the omega leader RNA and its experimental verification.

(a) Scheme of 5′-terminal blockade of the omega leader RNA with the synthetic oligodeoxyribonucleotide (see the text), which forms a stable untranslatable hairpin involving the 5′-end of the RNA. Black letters: the polyribonucleotide (RNA) comprising 67 nt long omega leader sequence (underlined); red letters: the blocking 55 nt long oligodeoxyribonucleotide containing SmaI restriction site (underlined). The arrows indicate the targets for restriction endonuclease SmaI and RNase H. The right-hand arrow at the end of the UTR sequence shows the start of the170 nt long RNA fragment encoding for the N-terminal part of firefly luciferase. The non-blocked mRNA construct consists of 254 nt and the ligation procedure yields a mixed polydeoxyribo-polyribonucleotide product of 309 nt in length. (b, c, d) Electrophoretic analyses of the polynucleotide preparations in denaturing 6% PAGE. (b) Analysis of the ligation reaction products: 1, standard RNA markers (100–1,000 nt); 2, the result of the ligation reaction of the RNA with the blocking oligodeoxyribonucleotide (the upper band corresponds to the blocked RNA); 3, the initial unblocked RNA. (c) Analysis of the blocked RNA after purification in PAGE: 1, unblocked RNA; 2, purified blocked RNA ligated with oligodeoxyribonucleotide. (d) Analysis of specificity of the ligation reaction product: 1, RNA markers (the same as in B); 2, the RNA blocked with the oligodeoxyribonucleotide; 3, the blocked RNA after treatment with endonuclease SmaI; 4, the blocked RNA after treatment with RNase H; 5, the unblocked RNA; 6, the unbloched RNA after treatment with endonuclease SmaI; 7, the unblocked RNA after treatment with RNase H.

Figure 2. Time-course curves of protein synthesis in cell-free translation systems based on wheat germ extract (WGE) (a) and rabbit reticulocyte lysate (RRL) (b).

Solid red curves: translation of the unblocked mRNA (5′UTR_omega-Luc₁₇₀3′); dashed blue curves: translation of the 5′-end blocked mRNA (5′DNA₅₅-UTR_omega-Luc₁₇₀3′).

Figure 3. Formation of ribosomal 48S initiation complexes at initiation AUG codon resulting from scanning of 5′-UTRs (coding regions of the mRNAs are not shown).

In each panel (a, b, c and d) the upper electrophoregram shows the result of incubation of mRNA with initiating ribosomal particles in the presence of the full set of initiation factors (eIF1, eIF1A, eIF2, eIF3, eIF4A, eIF4B, and eIF4F, together with initiator Met-tRNA_i and ATP), and the lower one demonstrates the result of the omission of eIF4F. The middle electrophoregram in panel (c) demonstrates the result of the omission of ATP. (a) Capped β-globin mRNA as a control. (b) Omega leader with capped 5′-end. (c) Omega leader with free (uncapped) 5′-end. (d) Omega leader with blocked 5′-end. Relative fluorescence intensities of cDNA products generated by reversed transcription are plotted against nucleotides of the leader sequences of corresponding mRNAs. On each electrophoregram the integral fluorescence of the left-hand major peaks reflects the amount of the full-length product when mRNA was read out by reversed transcription up to the 5′-end without stop. The integral fluorescence of the right-hand major peaks, here described as a “trident”, corresponds to the product of the reversed transcription stopped by the initiation 48S ribosomal complex formed at the initiation AUG codon.

Figure 4. Effect of the eIF4A (R362Q) mutant protein on the formation of ribosomal 48S initiation complexes at initiation AUG codon.

The upper electrophoregram in each panel (a, b) shows the result of incubation of mRNA with ribosomal particles and the full set of initiation factors (for more details see the legend to Fig. 3), and the lower one demonstrates the result of the addition of the eIF4A (R362Q) mutant (0.1 mg/ml). (a) Capped β-globin mRNA as a control. (b) Omega leader with uncapped 5′-end.

Figure 5. Effect of the eIF4A (R362Q) mutant on the time-course curves of luciferase (Luc) synthesis in cell-free translation system based on rabbit reticulocyte lysate (RRL).

(a): Translation of Luc-mRNA with capped β-globin leader. (b): Translation of Luc-mRNA with uncapped omega leader. Blue curves: control experiments (no eIF4A (R362Q) is added); red curves: translation in the presence of the eIF4A (R362Q) mutant (0.1 mg/ml). The luciferase synthesis was carried out and recorded in a luminometer cell in the presence of 0.2 mM luciferin and the luminescence was recorded online.