O6-Methylguanosine leads to position-dependent effects on ribosome speed and fidelity

Abstract

Nucleic acids are under constant assault from endogenous and environmental agents that alter their physical and chemical properties. O6-methylation of guanosine (m 6 G) is particularly notable for its high mutagenicity, pairing with T, during DNA replication. Yet, while m 6 G accumulates in both DNA and RNA, little is known about its effects on RNA. Here, we investigate the effects of m 6 G on the decoding process, using a reconstituted bacterial translation system. m 6 G at the first and third position of the codon decreases the accuracy of tRNA selection. The ribosome readily incorporates near-cognate aminoacyltRNAs (aa-tRNAs) by forming m 6 G-uridine codon-anticodon pairs. Surprisingly, the introduction of m 6 G to the second position of the codon does not promote miscoding, but instead slows the observed rates of peptide-bond formation by >1000-fold for cognate aa-tRNAs without altering the rates for near-cognate aa-tRNAs. These in vitro observations were recapitulated in eukaryotic extracts and HEK293 cells. Interestingly, the analogous modification N6-methyladenosine (m 6 A) at the second position has only a minimal effect on tRNA selection, suggesting that the effects on tRNA selection seen with m 6 G are due to altered geometry of the base pair. Given that the m6G:U base pair is predicted to be nearly indistinguishable from a WatsonCrick base pair, our data suggest that the decoding center of the ribosome is extremely sensitive to changes at the second position. Our data, apart from highlighting the deleterious effects that these adducts pose to cellular fitness, shed new insight into decoding and the process by which the ribosome recognizes codon-anticodon pairs.

How do cells cope with RNA damage and its consequences?

“…Similarly, on the ribosome, O6-mG causes miscoding by mispairing with U but only at the first position of the codon. At the second position, the adduct was found to reduce peptide-bond formation by almost 3 orders of magnitude (82). Again, these observations highlight not only the differences between the ribosome and DNA polymerases but also the position-dependent effects of these modifications on decoding.…”

“…In contrast to RNA oxidation, which has been investigated by a number of groups, only a couple of studies have explored the effect of alkylation damage to mRNA on translation. In one study from our group, we examined the effect of O6-mG on decoding using a bacterial reconstituted system (82). This adduct is notable as it is highly mutagenic during DNA replication, whereby the O6-mG:T bp is almost indistinguishable from a normal Watson-Crick bp such that mismatch repair is unable to recognize it (57).…”

Quality control of chemically damaged RNA

“…Specifically, some modifications can prevent base pairing completely, while others alter the base-pairing properties of the modified nucleotide. Consistent with these ideas are recent discoveries showing that chemically damaged RNAs pose significant hurdles to translational fidelity and efficiency [3, 4]. Modifications interfere with the decoding process on the ribosome, whereby codon–anticodon interactions are disrupted and, depending on the type of damage, result in stalling or miscoding.…”

“…It turns out that the effects of O6 Me G are due to inhibition of the GTPase activation step by elongation factor EF-Tu, a key step in the early phase of tRNA selection. Interestingly, the related modified nucleotide N 6 -methyladenosine (m 6 A) has only a modest effect on decoding when placed at the second position of the codon, suggesting that the effects on tRNA selection are not merely due to the introduction of a methyl group but rather due to altered geometry of the base pair [3]. This strongly suggests that the decoding center of the ribosome is extremely sensitive to changes to the second position of the codon–anticodon.…”

Regulation of DNA Alkylation Damage Repair: Lessons and Therapeutic Opportunities

“…Whether cells have the capacity to make this distinction between physiological versus alkylating agent-induced 1meA is not clear. The induction of 1meA by an alkylating agent likely induces this modification in an inappropriate region of an mRNA, potentially leading to inhibition of translation or ribosomal miscoding, a phenomenon previously demonstrated for O 6 meG in mRNA [94]. We term this blurring between what is considered damage-induced methylation versus physiological or epigenetic methylation as ‘epigenetic confusion’ (Figure 2A).…”