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. 2006 Apr;26(8):3164-9.
doi: 10.1128/MCB.26.8.3164-3169.2006.

A leaderless mRNA can bind to mammalian 80S ribosomes and direct polypeptide synthesis in the absence of translation initiation factors

Dmitri E Andreev  1 , Ilya M Terenin, Yan E Dunaevsky, Sergei E Dmitriev, Ivan N Shatsky
Affiliations

A leaderless mRNA can bind to mammalian 80S ribosomes and direct polypeptide synthesis in the absence of translation initiation factors

Dmitri E Andreev et al. Mol Cell Biol. 2006 Apr.

Abstract

Translation initiation in eukaryotic cells is known to be a complex multistep process which involves numerous protein factors. Here we demonstrate that leaderless mRNAs with initiator Met-tRNA can bind directly to 80S mammalian ribosomes in the absence of initiation factors and that the complexes thus formed are fully competent for the subsequent steps of polypeptide synthesis. We show that the canonical 48S pathway of eukaryotic translation initiation has no obvious advantage over the 80S pathway of translation initiation on leaderless mRNAs and suggest that, in the presence of competing mRNAs containing a leader, the latter mechanism will be preferred. The direct binding of the leaderless mRNA to the 80S ribosome was precluded when such an mRNA was supplied with a 5' leader, irrespective of whether it was in a totally single-stranded conformation or was prone to base pairing. The striking similarity between the mechanisms of binding of leaderless mRNAs with mammalian 80S or bacterial 70S ribosomes gives support to the idea that the alternative mode of translation initiation used by leaderless mRNAs represents a relic from early steps in the evolution of the translation apparatus.

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Figures

FIG. 1.
FIG. 1.
Formation of the ternary complex 80S ribosome-cIlacZ mRNA-Met-formula image. (A) Sucrose gradient sedimentation of the incubation mixtures of the cI mRNA and Met-formula image with mammalian 40S ribosomal subunits or 80S ribosomes. (B) The toeprint analysis of the same mixtures. Lanes 5 and 6 show toeprinting for mutant cIlacZ mRNAs where the initiation AUG codon was replaced by the GUG and UAA triplets, respectively. The 5′-terminal sequence of cIlacZ mRNA is shown at the bottom of the figure. Positions of toeprint bands are indicated by asterisks.
FIG. 2.
FIG. 2.
Elongation of the polypeptide by the 80S ribosome-cI mRNA-Met-formula image complex assembled without translation initiation factors. Positions of the toeprint bands originated from the AUG initiation codon and the triplet (AAA) preceding the termination codon are shown on the left of the gel. A dideoxynucleotide sequence generated with the same primer was run in parallel (shown on the right of the gel). The full-length product of primer extension is denoted “FL.” The sequence of the mutant cI(UAA-7) mRNA is presented below the autograph. The termination nucleotide triplet is underlined, and the initiation codon and the codon preceding the termination one are shown in boldface. Asterisks above the sequence show positions of the toeprint bands.
FIG. 3.
FIG. 3.
Effect of translation initiation factors on the formation of 48S translation initiation complexes. The molar concentrations of 40S ribosomes and cIlacZ mRNA used in these assays were exactly the same as those in the experiments on formation of 80S ribosome-cI mRNA-Met-formula image complexes. The yield of 48S initiation complexes was estimated as described in Materials and Methods and is presented below the toeprints. The values represent the average from three independent assembly experiments. For other designations, see the legends to Fig. 1 and 2.
FIG. 4.
FIG. 4.
Translation of the leaderless cIlacZ mRNA and leadered mRNAs possessing the same coding sequence in RRL. (A) Effect of capping of the leaderless and leadered transcripts on their translational efficiency. Constructs caa-cIlacZ and bAct-cIlacZ contained the (CAA)19 5′ leader and the 5′-UTR from the beta-actin mRNA, respectively. (B) Effect of mRNA concentration in the cell-free system on the translation efficiency of cIlacZ mRNA and a capped mRNA where the 5′UTR of beta-actin mRNA was fused to the cIlacZ coding sequence (capbAct-cIlacZ). (C) Effect of the PKR activity induced in RRL by incubation with the dsRNA on the translation of the cIlacZ mRNA and the capbAct-cIalcZ mRNA. Signs “+” and “++” correspond to the additions of 10 and 100 ng of dsRNA per 10 μl of the translation mixture, respectively. (D and E) dsRNA-induced phosphorylation of eIF2α. (D) Electrophoresis of proteins eluted from anti-eIF2 adsorbents. The gel was stained with Coomassie blue. “H” and “L” denote the heavy and light chains of immunoglobulin G. (E) The central and right lanes of the gel shown in panel D but developed with PhosphorImager. For other experimental details, see Materials and Methods.
FIG. 5.
FIG. 5.
Effect of 5′-UTR additions and a nucleotide context of the AUG codon of cIlacZ mRNA on its ability to directly program 80S ribosomes. Abbreviated names of RNA constructs are shown above the lanes. The same cIlacZ coding sequence was linked to the beta-actin 5′-UTR (bAct-cIlacZ), unstructured (CAA)19 leader (caa-cIlacZ), and short leaders GGGCCGAUG (G4C2-cIlacZ) or GAAAAGAUG (A4G2-cIlacZ). The full-length products for the first two constructs are situated in the gel much above those for other transcripts. For space limitations, they are not shown in the figure.
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