title: Analysis of Nonsense-Mediated mRNA Decay at the Single-Cell Level Using Two Fluorescent Proteins.
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.
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia; Nizhny Novgorod State Medical Academy, Nizhny Novgorod, Russia.
authors.forename: N G
authors.initials: NG
authors.lastname: Gurskaya
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia.
authors.forename: A P
authors.initials: AP
authors.lastname: Pereverzev
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia.
authors.forename: D B
authors.initials: DB
authors.lastname: Staroverov
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia.
authors.forename: N M
authors.initials: NM
authors.lastname: Markina
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia; Nizhny Novgorod State Medical Academy, Nizhny Novgorod, Russia. Electronic address: kluk@ibch.ru.
authors.forename: K A
authors.initials: KA
authors.lastname: Lukyanov
mesh_terms: D002460:Cell Line; D018389:Codon, Nonsense / Q000235:genetics; D005434:Flow Cytometry / Q000379:methods; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics*; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics*; D008856:Microscopy, Fluorescence / Q000379:methods; D059365:Nonsense Mediated mRNA Decay; D012333:RNA, Messenger / Q000235:genetics*; D059010:Single-Cell Analysis / Q000379:methods*; D000097573:Red Fluorescent Protein
chemical_list: D018389:Codon, Nonsense; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D012333:RNA, Messenger; D000097573:Red Fluorescent Protein
keywords: Cell heterogeneity; Flow cytometry; GFP; NMD; Premature termination codon; RFP; mRNA stability