A system for coordinated analysis of translational readthrough and nonsense-mediated mRNA decay

PLoS One. 2017 Mar 21;12(3):e0173980. doi: 10.1371/journal.pone.0173980. eCollection 2017.

Abstract

The nonsense-mediated mRNA decay (NMD) pathway degrades mRNAs containing premature termination codons, limiting the expression of potentially deleterious truncated proteins. This activity positions the pathway as a regulator of the severity of genetic diseases caused by nonsense mutations. Because many genetic diseases result from nonsense alleles, therapeutics inducing readthrough of premature termination codons and/or inhibition of NMD have been of great interest. Several means of enhancing translational readthrough have been reported to concomitantly inhibit NMD efficiency, but tools for systematic analysis of mammalian NMD inhibition by translational readthrough are lacking. Here, we introduce a system that allows concurrent analysis of translational readthrough and mRNA decay. We use this system to show that diverse readthrough-promoting RNA elements have similar capacities to inhibit NMD. Further, we provide evidence that the level of translational readthrough required for protection from NMD depends on the distance of the suppressed termination codon from the end of the mRNA.

MeSH terms

  • Base Sequence
  • Cell Line, Tumor
  • Codon, Nonsense / genetics*
  • Colorado tick fever virus / genetics
  • Epidermolysis Bullosa / genetics
  • Genetic Diseases, Inborn / genetics
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • Inverted Repeat Sequences / genetics
  • Moloney murine leukemia virus / genetics
  • Nonsense Mediated mRNA Decay / genetics*
  • Promoter Regions, Genetic / genetics
  • Protein Biosynthesis / genetics*
  • RNA Stability / genetics*
  • RNA, Messenger / metabolism*

Substances

  • Codon, Nonsense
  • RNA, Messenger

Grant support

This work was supported by the Intramural Research Program, National Institutes of Health, National Heart, Lung, and Blood Institute. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.