Nonsense-mediated mRNA decay uses complementary mechanisms to suppress mRNA and protein accumulation.

Dylan B Udy,Robert K Bradley

doi:10.26508/lsa.202101217

Abstract

Nonsense-mediated mRNA decay (NMD) is an essential, highly conserved quality control pathway that detects and degrades mRNAs containing premature termination codons. Although the essentiality of NMD is frequently ascribed to its prevention of truncated protein accumulation, the extent to which NMD actually suppresses proteins encoded by NMD-sensitive transcripts is less well-understood than NMD-mediated suppression of mRNA. Here, we describe a reporter system that permits accurate quantification of both mRNA and protein levels via stable integration of paired reporters encoding NMD-sensitive and NMD-insensitive transcripts into the AAVS1 safe harbor loci in human cells. We use this system to demonstrate that NMD suppresses proteins encoded by NMD-sensitive transcripts by up to eightfold more than the mRNA itself. Our data indicate that NMD limits the accumulation of proteins encoded by NMD substrates by mechanisms beyond mRNA degradation, such that even when NMD-sensitive mRNAs escape destruction, their encoded proteins are still effectively suppressed.

Highlights

Nonsense-mediated mRNA decay (NMD) is a eukaryotic cellular surveillance system that acts to prevent the accumulation of potentially deleterious truncated proteins by targeting mRNAs with premature termination codons (PTCs) for degradation
We have developed a robust NMD reporter system for making precise, quantitative mRNA and protein level measurements (Fig 1)
This system builds on previous reporters and adds numerous features, including (1) luciferase domains for high dynamic range protein-level measurements, (2) internal control reporters for accurate normalization across samples, (3) dox inducibility for mRNA stability measurements, and (4) stable integration at the AAVS1 safe harbor loci for predictable genomic integration and uniform expression

Summary

Introduction

Nonsense-mediated mRNA decay (NMD) is a eukaryotic cellular surveillance system that acts to prevent the accumulation of potentially deleterious truncated proteins by targeting mRNAs with premature termination codons (PTCs) for degradation (seminal articles: Chang et al [1979], Losson and Lacroute [1979], Maquat et al [1981], and Kinniburgh et al [1982]; reviewed in: Lykke-Andersen and Jensen [2015] and Kurosaki et al [2019]). Truncated proteins derived from NMD-insensitive transcripts, in which the PTC resides in the last exon or last ~55 nucleotides of the penultimate exon, can cause disease in heterozygotes, whereas heterozygous individuals bearing PTCs that generate NMD-sensitive transcripts in the same genes are often unaffected (Holbrook et al, 2004; Khajavi et al, 2006; Miller & Pearce, 2014; Coban-Akdemir et al, 2018) These genetic findings strongly support the hypothesis that limiting potentially deleterious truncated protein accumulation is essential for cell health and homeostasis and likely one of the primary selection pressures for evolution and maintenance of the NMD pathway. In the absence of highly quantitative proteinlevel measurements, the extent to which protein versus mRNA alone is suppressed remains unclear

Methods

Results

Conclusion