Analysis of nonsense-mediated mRNA decay in mammalian cells

Abstract

The nonsense-mediated mRNA decay (NMD) pathway acts to selectively identify and degrade mRNAs that contain a premature translation termination codon (PTC), and hence reduce the accumulation of potentially toxic truncated proteins. NMD is one of the best studied mRNA quality-control mechanisms in eukaryotes, and it has become clear during recent years that many physiological mRNAs are also NMD substrates, signifying a role for NMD beyond mRNA quality control as a translation-dependent post-transcriptional regulator of gene expression. Despite a great deal of scientific research for over twenty years, the process of NMD is far from being fully understood with regard to its physiological relevance to the cell, the molecular mechanisms that underpin this pathway, all of the factors that are involved, and the exact cellular locations of NMD. This unit details some of the fundamental RNA based approaches taken to examine aspects of NMD, such as creating PTC+ reporter genes, knocking down key NMD factors via RNAi, elucidating the important functions of NMD factors by complementation assays or Tethered Function Assays, and measuring RNA levels by reverse-transcription quantitative PCR.