The bacterial toxin RelE induces specific mRNA cleavage in the A site of the eukaryote ribosome

Abstract

RelE/RelB is a well-characterized toxin-anti-toxin pair involved in nutritional stress responses in Bacteria and Archae. RelE lacks any eukaryote homolog, but we demonstrate here that it efficiently and specifically cleaves mRNA in the A site of the eukaryote ribosome. The cleavage mechanism is similar to that in bacteria, showing the feasibility of A-site cleavage of mRNA for regulatory purposes also in eukaryotes. RelE cleavage in the A-site codon of a stalled eukaryote ribosome is precise and easily monitored, making "RelE printing" a useful complement to toeprinting to determine the exact mRNA location on the eukaryote ribosome and to probe the occupancy of its A site.

FIGURE 1.

RelE induces cleavage of β-globin mRNA within 48S initiation complexes formed in RRL Positions of toeprint and RelE-print bands are shown on the right of the gel. A dideoxynucleotide sequence generated with the same primer was run in parallel (shown on the left of the gel). The full-length product of primer extension is denoted as “FL.” Positions of bands with respect to the A-residue of the AUG initiation codon are shown on the right of the gel. Lane 1 corresponds to the negative control (Mg²⁺ was added to final concentration 30 mM prior to addition of mRNA to prevent initiation complex formation).

FIGURE 2.

RelE induces cleavage of β-globin mRNA in the 48S (A) and 80S (B) initiation complexes generated from purified components. Positions of toeprint and RelE-print bands are indicated at the opposite sides of each gel. The full-length product of primer extension is denoted as “FL.”

FIGURE 3.

RelE printing and toeprinting of 40S·mRNA binary complex and 48S complex formed on the HCV mRNA (A), 80S·Met-tRNAi·c1 lacZ mRNA complex (B), and the pretermination complex formed on c1(UAA-7)lacZ mRNA (C). For A and B, the positions of the toeprint and RelE-print bands are indicated on the opposite sides of each gel. For C, positions of the toeprint bands originating from complexes at the AUG initiation codon and the triplet (AAA) preceding the termination codon and RelE-print bands 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 triplet preceding the termination signal are shown in boldface. Asterisks above the sequence show the positions of the toeprint bands. Empty arrows show the positions of the RelE-induced cleavages.

FIGURE 4.

(A) RelE printing and toeprinting of HRV-Fluc and FMDV-Fluc RNAs in the combined RRL-HeLa extracts. cDNA ladders for these RNAs are shown on the left and right of the gel, respectively. Groups of lanes marked “extension” show the results of toeprinting, whereas those denoted as “deproteinization extension” correspond to RelE-print assays. For comparison, the assays with RRL only also presented (lanes 1,2,4,5,8,9,11,12). Lanes 1 and 8 are control assays where the reconstitution was blocked by 30 mM of magnesium. (B) RelE-induced cleavage of the β-globin mRNA in 48S complexes formed on near-cognate codons in the absence of eIF1. The translation initiation complexes on the β-globin mRNA were assembled under the same conditions and with the same batches of components as in the experiments shown in Fig. 2 except eIF1 was not added to the reconstitution system. For higher resolution of upper bands, a long run gel was required (2 h 10 min instead of regular 1 h 10 min), and therefore, the coding part of the mRNA is not presented in the gel. The positions of toeprint and RelE-print bands as well as the sequence of the β-globin mRNA are shown on the right of the gel. The sequence of the β-globin mRNA is also presented on the right of the autograph. Lane 1 shows results of toeprinting in the absence of eIF1. Lanes 2 and 3 are the primer extensions on free mRNAs isolated from reconstitution mixtures treated (+) and untreated (−) with RelE. The position of AUG triplet and near-cognate triplets are shown in boxes. Empty arrows show the positions of RelE attack. Asterisks denote the position of the initiation codon.