NMD monitors translational fidelity 24/7

Abstract

Nonsense-mediated mRNA decay (NMD) is generally thought to be a eukaryotic mRNA surveillance pathway tasked with the elimination of transcripts harboring an in-frame premature termination codon (PTC). As presently conceived, NMD acting in this manner minimizes the likelihood that potentially toxic polypeptide fragments would accumulate in the cytoplasm. This notion is to be contrasted to the results of systematic RNA-Seq and microarray analyses of NMD substrates in multiple model systems, two different experimental approaches which have shown that many mRNAs identified as NMD substrates fail to contain a PTC. Our recent results provide insight into, as well as a possible solution for, this conundrum. By high-resolution profiling of mRNAs that accumulate in yeast when the principal NMD regulatory genes (UPF1, UPF2, and UPF3) are deleted, we identified approximately 900 NMD substrates, the majority of which are normal-looking mRNAs that lack PTCs. Analyses of ribosomal profiling data revealed that the latter mRNAs tended to manifest elevated rates of out-of-frame translation, a phenomenon that would lead to premature translation termination in alternative reading frames. These results, and related observations of heterogeneity in mRNA isoforms, suggest that NMD should be reconsidered as a probabilistic mRNA quality control pathway that is continually active throughout an mRNA's life cycle.

Keywords: Frameshifting; NMD substrates; Probabilistic mRNA decay; Translational fidelity.

Fig. 1

Ribosomal failure to maintain the correct mRNA reading frame is a common basis for NMD targeting. The figure depicts translation of a “normal-looking” mRNA and the ribosomal elongation events that lead to its acquisition of NMD substrate status. Top: blue ribosomes are translating the mRNA in the annotated open reading frame (gray and black boxes) whereas the pink ribosomes have entered a stretch of non-optimal codons (pink, orange, and red boxes) and shifted to the +1 reading frame. Bottom: higher resolution depiction of a premature termination event that occurs as a consequence of ribosomal elongation in the +1 reading frame

Fig. 2

NMD targets distinct types of substrates. mRNAs become substrates for NMD when an elongating ribosome encounters a PTC that has been generated in one of several different ways. a “Traditional” NMD substrates. For these mRNAs, translation begins at initiation codons located at ORF (or uORF) 5′ ends, and elongation then proceeds 3′, leading to ribosomal A site positioning of an in-frame PTC. Substrates in this class include mRNAs derived from nonsense alleles, intron-containing pre-mRNAs that enter the cytoplasm, uORF-containing mRNAs, mRNAs in which programmed frameshifting allows a fraction of ribosomes to avoid premature termination, and mRNAs transcribed from pseudogenes. b “Probabilistic” NMD substrates. These mRNAs lack in-frame PTCs in their annotated ORFs, but contain features that promote either downstream out-of-frame translational initiation or frameshifting, thus leading to premature translation termination in a new reading frame. mRNAs in this category can have poor sequence context around the normal initiation codon, a transcription start site that is internal to the principal ORF, lower overall codon optimality, or a long stretch of non-optimal codons (NOCs). In each example, a subset of ribosomes translates the mRNA in a frame different from that of the annotated ORF. Green initiation codon; red stop codon; yellow UTR; purple stop codon encountered in the +1 or +2 reading frame; blue cluster of non-optimal codons. From Celik et al. (2017)