Oxidative stress damages rRNA inside the ribosome and differentially affects the catalytic center

Abstract

Intracellular levels of reactive oxygen species (ROS) increase as a consequence of oxidative stress and represent a major source of damage to biomolecules. Due to its high cellular abundance RNA is more frequently the target for oxidative damage than DNA. Nevertheless the functional consequences of damage on stable RNA are poorly understood. Using a genome-wide approach, based on 8-oxo-guanosine immunoprecipitation, we present evidence that the most abundant non-coding RNA in a cell, the ribosomal RNA (rRNA), is target for oxidative nucleobase damage by ROS. Subjecting ribosomes to oxidative stress, we demonstrate that oxidized 23S rRNA inhibits the ribosome during protein biosynthesis. Placing single oxidized nucleobases at specific position within the ribosome's catalytic center by atomic mutagenesis resulted in markedly different functional outcomes. While some active site nucleobases tolerated oxidative damage well, oxidation at others had detrimental effects on protein synthesis by inhibiting different sub-steps of the ribosomal elongation cycle. Our data provide molecular insight into the biological consequences of RNA oxidation in one of the most central cellular enzymes and reveal mechanistic insight on the role of individual active site nucleobases during translation.

Figure 1.

Oxidative stress to the ribosome disrupts translation and damages ribosomal RNA. (A) Structures of the most stable oxidation products found in RNA: 8-oxo-7,8-dihydroadenosine (8-oxo-A), 8-oxo-7,8-dihydroguanosine (8-oxo-G), 5-hydroxyuridine (5-OH-U), 5-hydroxycytidine (5-OH-C), and the abasic site (aba). The oxidations are highlighted in red. (B–D) In vitro translation activity of ribosomal components previously oxidized in vitro with increasing stress conditions (10 μM Fe(II)ascorbate and 0.1–20 mM H₂O₂). Translation of mRNA coding for r-protein L12 was tested by separating the [³⁵S]-labeled products by SDS-PAGE and autoradiography. (B) In vitro translation with oxidized E. coli 70S ribosomes (70S_ox). (C) Translation with oxidized T. aquaticus 50S subunits (50S_ox) complemented with unstressed E. coli 30S subunits. (D) Translation with oxidized E. coli 30S subunits (30S_ox) complemented with native E. coli 50S subunits. In (B), (C) and (D) complete translation reactions containing gradient-purified E. coli 30S or 50S ribosomal subunits, respectively, served as negative controls. The minor L12 product bands in these controls originate from minute amounts of contaminating 50S or 30S particles in the respective subunit preparation. (E) Translation with non-oxidized 30S subunits combined with reconstituted T. aquaticus 50S particles containing unstressed r-proteins and oxidized 23S rRNA (23S_ox). The negative control (30S) contained unstressed E. coli 30S subunits and complete 50S reconstitution samples but lacking 23S rRNA. (F) Translation with non-oxidized 50S subunits combined with reconstituted E. coli 30S particles containing unstressed r-proteins and oxidized 16S rRNA (16S_ox). The negative control (50S) contained unstressed E. coli 50S subunits and complete 30S reconstitution samples but lacking rRNA. The agarose gels at the bottom in (E) and (F) depict the integrity of the 23S and 16S rRNA, respectively, used for subunit reconstitution after increasing exposure to oxidative stress. (G) Quantification of relative translation activities with oxidized 50S or oxidized 30S subunits, and subunits reconstituted from oxidized 23S or 16S rRNA. Data shown represent the mean (n = 3) at the endpoints at the highest H₂O₂ levels in experiments shown in (C) through (F), normalized to translation with unstressed ribosomal subunits. Error bars indicate standard deviation.

Figure 2.

Oxidation in the ribosome's active site differentially affects protein synthesis. (A) Endpoint measurements in poly(Phe) synthesis employing in vitro reconstituted ribosomes containing either synthetic RNA oligonucleotides with the wildtype sequence (wt) in the active site, or oligos harboring specific nucleobase oxidations in the PTC or nearby regions. In all reconstituted cp-23S rRNA constructs the minute translational activities of ribosomes containing no oligo was subtracted as background, and the activity of ribosomes containing the corresponding wt oligonucleotide was taken as 1.00. Values represent the mean of at least three independent experiments, with SD shown as error bars. Significance according to paired Student's t-test: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. (B) Representative autoradiogram of an SDS-PAGE showing in vitro translation of L12 r-protein mRNA by reconstituted ribosomes carrying 8-oxo-adenosine at 23S rRNA position A2451 or 8-oxo-guanosine G2447. Reactions with ribosomes reconstituted without a complementing synthetic RNA oligo (–) served as negative control. (C) EF-G GTPase activation as measured by the rate of GTP hydrolysis using reconstituted ribosomes with the wt sequence (filled symbols) or oxidized PTC bases (open symbols). Values represent the mean of at least two independent time course experiments, with error bars indicating SD. (D) Translocation of ribosomes oxidized at position A2451 along a heteropolymeric mRNA as determined by toeprinting. Toeprint bands indicate the positions of mRNA-bound ribosome complexes before (PRE) and after (POST) addition of EF-G and GTP. Ribosomal complexes carried deacylated tRNA and peptidyl-tRNA in the P- and A-site in the PRE state, and in the E- and P-site in the POST complexes, respectively. Reactions in the presence of T. aquaticus 30S subunits alone served as negative controls. (E) Time course of peptide bond formation in ribosomes containing either the wt sequence or 8-oxo-A2451. Values represent the mean of 3 independent time course experiments. The activities of in vitro assembled ribosomes without a synthetic oligo were subtracted as background. The endpoint value of ribosomes containing the wt sequence was taken as 1.00. (F) SHAPE probing of the 8-oxo-A2451 oligo inside the reconstituted large ribosomal subunit. Reconstituted 50S were treated with NMIA to determine ribose 2′-OH reactivity, which was detected by primer extension analysis. The location of the radiolabeled primer, the full length reverse transcription product and the position of A2451 in the denaturing polyacrylamide gel are indicated by arrow heads and an arrow, respectively. Note that the NMIA reaction product halts reverse transcription one position 3′ of the reacted site. Primer extension reaction in the absence of any RNA was used as negative control (no RNA).

Figure 3.

Effects of oxidation at positions C2063, U2585 and A2602. (A) In vitro translation of r-protein L12 mRNA with chemically engineered ribosomes oxidized at position C2063. Reaction with ribosomes reconstituted without a complementing synthetic RNA oligo (–) served as negative control. (B) Time course of translocation of ribosomes oxidized at position C2063 along a heteroplymeric mRNA as determined by toeprinting. The translocation reaction was initiated by the addition of EF-G•GTP and stopped by the antibiotic neomycin at the indicated time points. The mean and standard deviation of three independent time course experiments are shown. (C) The non-Watson-Crick base pair formed in the 50S subunit between C2063 and A2450 is shown here with 5-OH-C (bold) in position 2063. The integrity of this interaction was shown previously to be vital for effective translation (40). (D) In vitro translation of r-protein L12 mRNA with chemically engineered ribosomes oxidized at position U2585. Reaction with ribosomes reconstituted without a complementing synthetic RNA oligo (–) served as negative control. (E) Ac-[³H]Phe-tRNA^Phe binding affinity to the A- or P-site, respectively, in chemically engineered ribosomes oxidized at position U2585. Binding activities were normalized to ribosomes harboring the synthetic wt oligo, whereas unspecific tRNA binding to ribosomes containing no synthetic oligo was always subtracted as background. The mean and standard deviation of 5–7 independent binding experiments are shown. Paired Student's t-test: **P ≤ 0.01; ns, statistically not significant. (F) Release reaction as measured by P-site-bound f[³H]Met-tRNA^fMet hydrolysis after RF-1 (n = 2) or RF-2 (n = 4) addition to chemically engineered ribosomes carrying an 8-oxo-adenosine at position A2602. f[³H]Met-tRNA^fMet hydrolysis was normalized to wt activities and f[³H]Met release in ribosomes containing no oligo was subtracted as background.