Chemical modifications to mRNA nucleobases impact translation elongation and termination

Abstract

Messenger RNAs (mRNAs) serve as blueprints for protein synthesis by the molecular machine the ribosome. The ribosome relies on hydrogen bonding interactions between adaptor aminoacyl-transfer RNA molecules and mRNAs to ensure the rapid and faithful translation of the genetic code into protein. There is a growing body of evidence suggesting that chemical modifications to mRNA nucleosides impact the speed and accuracy of protein synthesis by the ribosome. Modulations in translation rates have downstream effects beyond protein production, influencing protein folding and mRNA stability. Given the prevalence of such modifications in mRNA coding regions, it is imperative to understand the consequences of individual modifications on translation. In this review we present the current state of our knowledge regarding how individual mRNA modifications influence ribosome function. Our comprehensive comparison of the impacts of 16 different mRNA modifications on translation reveals that most modifications can alter the elongation step in the protein synthesis pathway. Additionally, we discuss the context dependence of these effects, highlighting the necessity of further study to uncover the rules that govern how any given chemical modification in an mRNA codon is read by the ribosome.

Keywords: Kinetics; RNA modification; Translation; mRNA modification.

Fig. 1.

RNAs form the basis of the protein synthesis machinery. (A) Depiction of the basic components of the translational machinery, with the three central RNA species highlighted (rRNA, tRNA and mRNA). (B) The four chemical building blocks of RNA: adenosine (A), guanosine (G), cytidine (C) and uridine (U).

Fig. 2.

Modifications in mRNAs. (A) Modifications reported to be enzymatically incorporated into mRNAs. (B) Modifications that can be incorporated as a result of RNA damage. (C) Modifications not found naturally found in mRNAs that are either incorporated into mRNA vaccines (m¹Ψ) or have been used to probe translation termination.

Fig. 3.

Assessing protein synthesis. (A) Schematic of the steps in bacterial protein synthesis In the first step (initiation), initiation factors (IFs) help the 70S ribosome form on an AUG start codon with fMet-tRNA^fMet bound in the ribosome P site. During the elongation phase of translation, aminoacyl-tRNAs (aa-tRNAs) are brought into the ribosome A site by EF-Tu:GTP (initial binding). The aa-tRNA is then positioned properly in the A site during accommodation, and EF-Tu exits following GTP hydrolysis. The amino acid (or peptide) on the P site tRNA is then transferred to the aa-tRNA in the ribosome A site during peptidyl-transfer. After a new peptide bond is catalyzed by the ribosome, EF-G binds and moves (translocates) the ribosome and associated tRNAs to the next codon. The cycle of elongation continues until the ribosome reaches a stop codon (UAA, UGA or UAG), and release factors (RFs) bind to the A site to catalyze the hydrolysis of the complete polypeptide. (B) Common approaches used to study translation. The kinetic resolution and mechanistic detail possible to attain increases as with the purification level of the translation system (from cells to reconstituted translation).

Fig. 4.

Modification of nucleobase positions impacts translation. Positions in adenosine (A), uridine (B), guanosine (C) and cytidine (D) that impede only elongation rates (red), elongation rates and amino acid mis-incorporation (blue), translation termination (green) are circled. The data are too preliminary to confidently assign impacts to positions highlighted in orange. (For interpretation of the references to colour in this figure legend, the reader is referred to Table 1 and the web version of this article.)