A premature termination codon (PTC) in the ORF of an mRNA generally leads to production of a truncated polypeptide, accelerated degradation of the mRNA, and depression of overall mRNA expression. Accordingly, nonsense mutations cause some of the most severe forms of inherited disorders. The small-molecule drug ataluren promotes therapeutic nonsense suppression and has been thought to mediate the insertion of near-cognate tRNAs at PTCs. However, direct evidence for this activity has been lacking. Here, we expressed multiple nonsense mutation reporters in human cells and yeast and identified the amino acids inserted when a PTC occupies the ribosomal A site in control, ataluren-treated, and aminoglycoside-treated cells. We find that ataluren’s likely target is the ribosome and that it produces full-length protein by promoting insertion of near-cognate tRNAs at the site of the nonsense codon without apparent effects on transcription, mRNA processing, mRNA stability, or protein stability. The resulting readthrough proteins retain function and contain amino acid replacements similar to those derived from endogenous readthrough, namely Gln, Lys, or Tyr at UAA or UAG PTCs and Trp, Arg, or Cys at UGA PTCs. These insertion biases arise primarily from mRNA:tRNA mispairing at codon positions 1 and 3 and reflect, in part, the preferred use of certain nonstandard base pairs, e.g., U-G. Ataluren’s retention of similar specificity of near-cognate tRNA insertion as occurs endogenously has important implications for its general use in therapeutic nonsense suppression.

Chemical modifications of RNAs have long been established as key modulators of nonprotein-coding RNA structure and function in cells. There is a growing appreciation that messenger RNA (mRNA) sequences responsible for directing protein synthesis can also be posttranscriptionally modified. The enzymatic incorporation of mRNA modifications has many potential outcomes, including changing mRNA stability, protein recruitment, and translation. We tested how one of the most common modifications present in mRNA coding regions, pseudouridine (Ψ), impacts protein synthesis using a fully reconstituted bacterial translation system and human cells. Our work reveals that replacing a single uridine nucleotide with Ψ in an mRNA codon impedes amino acid addition and EF-Tu GTPase activation. A crystal structure of the Thermus thermophilus 70S ribosome with a tRNAPhe bound to a ΨUU codon in the A site supports these findings. We also find that the presence of Ψ can promote the low-level synthesis of multiple peptide products from a single mRNA sequence in the reconstituted translation system as well as human cells, and increases the rate of near-cognate Val-tRNAVal reacting on a ΨUU codon. The vast majority of Ψ moieties in mRNAs are found in coding regions, and our study suggests that one consequence of the ribosome encountering Ψ can be to modestly alter both translation speed and mRNA decoding.

Premature termination codons (PTCs) in an mRNA ORF inactivate gene function by causing production of a truncated protein and destabilization of the mRNA. Readthrough of a PTC allows ribosomal A-site insertion of a near-cognate tRNA, leading to synthesis of a full-length protein from otherwise defective mRNA. To understand the mechanism of such nonsense suppression, we developed a yeast system that allows purification and sequence analysis of full-length readthrough products arising as a consequence of endogenous readthrough or the compromised termination fidelity attributable to the loss of Upf (up-frameshift) factors, defective release factors, or the presence of the aminoglycoside gentamicin. Unlike classical "wobble" models, our analyses showed that three of four possible near-cognate tRNAs could mispair at position 1 or 3 of nonsense codons and that, irrespective of whether readthrough is endogenous or induced, the same sets of amino acids are inserted. We identified the insertion of Gln, Tyr, and Lys at UAA and UAG, whereas Trp, Arg, and Cys were inserted at UGA, and the frequency of insertion of individual amino acids was distinct for specific nonsense codons and readthrough-inducing agents. Our analysis suggests that the use of genetic or chemical means to increase readthrough does not promote novel or alternative mispairing events; rather, readthrough effectors cause quantitative enhancement of endogenous mistranslation events. Knowledge of the amino acids incorporated during readthrough not only elucidates the decoding process but also may allow predictions of the functionality of readthrough protein products.translation termination | nonsense codon readthrough | therapeutic nonsense suppression T he information in an mRNA ORF can be repurposed during translation by several different types of recoding events, including leaky scanning, initiation at noncanonical start codons, ribosome frame-shifting, or stop codon readthrough (1). Stop codon readthrough, or nonsense suppression, occurs when a ribosome positioned with a nonsense codon in its A-site incorporates an amino acid into the nascent polypeptide chain instead of terminating translation by promoting the hydrolysis and release of that polypeptide from the P-site peptidyl-tRNA (2, 3). Two release factors mediate termination in eukaryotes: eRF1 (eukaryotic release factor 1), a protein that directly recognizes the three nonsense codons (UAA, UAG, or UGA), and eRF3, a GTPase that binds eRF1 and stimulates its activity (2, 4). Although translation termination is the kinetically favored event when a nonsense codon occupies the ribosomal A-site, recognition of that nonsense codon by eRF1 is subject to competition from near-cognate aminoacyl-tRNAs (2). Productive binding of a near-cognate aminoacyl-tRNA to an A-site harboring a nonsense codon bypasses termination and results in stop codon readthrough. Readthrough of a normal termination codon (NTC) leads to translation elongation into the mRNA 3′-UTR, generating a C-terminally extended polypeptide, whereas ...

The decay rate of an mRNA and the efficiency with which it is translated are key determinants of eukaryotic gene expression. Although it was once thought that mRNA stability and translational efficiency were directly linked, the interrelationships between the two processes are considerably more complex. The decay of individual mRNAs can be triggered or antagonized by translational impairment, and alterations in the half-life of certain mRNAs can even alter translational fidelity. In this review, we consider whether mRNA translation and turnover are distinct or overlapping phases of an mRNA life cycle, and then address some of the many ways in which the two processes influence each other in eukaryotic cells.