title: Method for quantitative analysis of nonsense-mediated mRNA decay at the single cell level.
abstract: Nonsense-mediated mRNA decay (NMD) is a ubiquitous mechanism of degradation of transcripts with a premature termination codon. NMD eliminates aberrant mRNA species derived from sources of genetic variation such as gene mutations, alternative splicing and DNA rearrangements in immune cells. In addition, recent data suggest that NMD is an important mechanism of global gene expression regulation. Here, we describe new reporters to quantify NMD activity at the single cell level using fluorescent proteins of two colors: green TagGFP2 and far-red Katushka. TagGFP2 was encoded by mRNA targeted to either the splicing-dependent or the long 3'UTR-dependent NMD pathway. Katushka was used as an expression level control. Comparison of the fluorescence intensities of cells expressing these reporters and cells expressing TagGFP2 and Katushka from corresponding control NMD-independent vectors allowed for the assessment of NMD activity at the single cell level using fluorescence microscopy and flow cytometry. The proposed reporter system was successfully tested in several mammalian cell lines and in transgenic Xenopus embryos.
authors.affiliation: Institute of Bioorganic Chemistry, Miklukho-Maklaya 16/10, 117997 Moscow, Russia.
authors.forename: Anton P
authors.initials: AP
authors.lastname: Pereverzev
authors.affiliation: Institute of Bioorganic Chemistry, Miklukho-Maklaya 16/10, 117997 Moscow, Russia.
authors.forename: Nadya G
authors.initials: NG
authors.lastname: Gurskaya
authors.affiliation: Institute of Bioorganic Chemistry, Miklukho-Maklaya 16/10, 117997 Moscow, Russia.
authors.forename: Galina V
authors.initials: GV
authors.lastname: Ermakova
authors.affiliation: Institute of Bioorganic Chemistry, Miklukho-Maklaya 16/10, 117997 Moscow, Russia.
authors.forename: Elena I
authors.initials: EI
authors.lastname: Kudryavtseva
authors.affiliation: Institute of Bioorganic Chemistry, Miklukho-Maklaya 16/10, 117997 Moscow, Russia.
authors.forename: Nadezhda M
authors.initials: NM
authors.lastname: Markina
authors.affiliation: Institute of Bioorganic Chemistry, Miklukho-Maklaya 16/10, 117997 Moscow, Russia.
authors.forename: Alexey A
authors.initials: AA
authors.lastname: Kotlobay
authors.affiliation: 1] Institute of Bioorganic Chemistry, Miklukho-Maklaya 16/10, 117997 Moscow, Russia [2] Nizhny Novgorod State Medical Academy, Minin Sq. 10/1, 603005 Nizhny Novgorod, Russia.
authors.forename: Sergey A
authors.initials: SA
authors.lastname: Lukyanov
authors.affiliation: Institute of Bioorganic Chemistry, Miklukho-Maklaya 16/10, 117997 Moscow, Russia.
authors.forename: Andrey G
authors.initials: AG
authors.lastname: Zaraisky
authors.affiliation: 1] Institute of Bioorganic Chemistry, Miklukho-Maklaya 16/10, 117997 Moscow, Russia [2] Nizhny Novgorod State Medical Academy, Minin Sq. 10/1, 603005 Nizhny Novgorod, Russia.
authors.forename: Konstantin A
authors.initials: KA
authors.lastname: Lukyanov
mesh_terms: D020413:3' Untranslated Regions / Q000235:genetics; D000818:Animals; D004625:Embryo, Nonmammalian / Q000378:metabolism; D005434:Flow Cytometry; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008856:Microscopy, Fluorescence; D059365:Nonsense Mediated mRNA Decay / Q000235:genetics*; D012326:RNA Splicing / Q000235:genetics; D059010:Single-Cell Analysis / Q000379:methods*; D014982:Xenopus laevis
chemical_list: D020413:3' Untranslated Regions; D049452:Green Fluorescent Proteins