Modulation of translational decoding by m6A modification of mRNA

Abstract

N6-methyladenosine (m6A) is an abundant, dynamic mRNA modification that regulates key steps of cellular mRNA metabolism. m6A in the mRNA coding regions inhibits translation elongation. Here, we show how m6A modulates decoding in the bacterial translation system using a combination of rapid kinetics, smFRET and single-particle cryo-EM. We show that, while the modification does not impair the initial binding of aminoacyl-tRNA to the ribosome, in the presence of m6A fewer ribosomes complete the decoding process due to the lower stability of the complexes and enhanced tRNA drop-off. The mRNA codon adopts a π-stacked codon conformation that is remodeled upon aminoacyl-tRNA binding. m6A does not exclude canonical codon-anticodon geometry, but favors alternative more dynamic conformations that are rejected by the ribosome. These results highlight how modifications outside the Watson-Crick edge can still interfere with codon-anticodon base pairing and complex recognition by the ribosome, thereby modulating the translational efficiency of modified mRNAs.

Identifying N6-Methyladenosine Sites in HepG2 Cell Lines Using Oxford Nanopore Technology

“…However, there is evidence that m6A may also enhance translation [5]. The impact of these modifications depends on multiple factors, including the presence of specific m6A reader proteins and the location of m6A on the mRNA [5,[26][27][28].…”

Interconnections between m6A RNA modification, RNA structure, and protein–RNA complex assembly

“…stability and enhancing the tRNA drop-off. This consequently results in a smaller number of ribosomes that complete the decoding process, resulting in a less efficient translation of the affected transcripts (Jain et al, 2023).…”

“…Another study recently confirmed these findings by showing that m 6 A does not prevent canonical codon–anticodon base-pairing but favors alternative conformations leading to a lower stability and enhancing the tRNA drop-off. This consequently results in a smaller number of ribosomes that complete the decoding process, resulting in a less efficient translation of the affected transcripts ( Jain et al, 2023 ).…”

Atomic Mutagenesis of N6-Methyladenosine Reveals Distinct Recognition Modes by Human m6A Reader and Eraser Proteins

“…While the syn orientation is energetically preferred in the free nucleosides and in single-stranded RNA, formation of a standard Watson–Crick base pair requires the methyl group to rotate into the anti conformation. − Thus, m 6 A influences local structures and base pairing kinetics, which in turn affect global RNA folding, structures, and associated functions. Multiple studies have shown that m 6 A modulates the fate of mRNAs by influencing their stability, translation efficiency, and splicing through direct or indirect interaction with proteins. ,,, A group of direct m 6 A reader proteins each contain the YTH domain, which forms a hydrophobic binding pocket for m 6 A. Well-established human m 6 A readers include the proteins YTHDF1/DF2/DF3 and YTHDC1/DC2 . Although these YTH-domain containing proteins seem to bind m 6 A-containing RNA via similar mechanisms, they cause different downstream effects. , Interestingly, m 6 A can be reverted to adenosine through oxidative demethylation by the human m 6 A demethylase enzymes FTO and ALKBH5 .…”