Analysis of Nonsense-Mediated mRNA Decay at the Single-Cell Level Using Two Fluorescent Proteins

N. G. Gurskaya, A. P. Pereverzev, D. B. Staroverov, N. M. Markina, K. A. Lukyanov

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

5 Citations (Scopus)

Abstract

Nonsense-mediated mRNA decay (NMD) is an evolutionarily conserved mechanism of specific degradation of transcripts with a premature stop codon. NMD eliminates aberrant mRNAs arising from mutations, alternative splicing, and other events in cells. In addition, many normal transcripts undergo NMD. Recent studies demonstrated that NMD activity is specifically regulated and that NMD can play a role of global regulator of gene expression. Recently, we developed dual-color fluorescent protein-based reporters for quantification of NMD activity using fluorescence microscopy and flow cytometry (Pereverzev, Gurskaya, et al., 2015). Due to ratiometric fluorescence response, these reporters make it possible to assess NMD activity in live cells at the single-cell level and to reveal otherwise hidden heterogeneity of cells in respect of NMD activity. Here we provide a detailed description of applications of the NMD reporters in mammalian cell lines.

Original languageEnglish
Title of host publicationMethods in Enzymology
PublisherAcademic Press Inc.
Pages291-314
Number of pages24
DOIs
Publication statusPublished - 2016
Externally publishedYes

Publication series

NameMethods in Enzymology
Volume572
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Keywords

  • Cell heterogeneity
  • Flow cytometry
  • GFP
  • mRNA stability
  • NMD
  • Premature termination codon
  • RFP

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