Образование

Премии и заслуги

Премия имени Р.Б. Хесина 1 степени (2012)

Гранты и проекты

Избранные публикации

1. Gurskaya N.G., Pereverzev A.P., Staroverov D.B., Markina N.M., Lukyanov K.A. (2016). Analysis of Nonsense-Mediated mRNA Decay at the Single-Cell Level Using Two Fluorescent Proteins. Meth. Enzymol. 572, 291–314 [+]

   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.

   ID:1528
2. Pereverzev A.P., Gurskaya N.G., Ermakova G.V., Kudryavtseva E.I., Markina N.M., Kotlobay A.A., Lukyanov S.A., Zaraisky A.G., Lukyanov K.A. (2015). Method for quantitative analysis of nonsense-mediated mRNA decay at the single cell level. Sci Rep 5, 7729 [+]

   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.

   ID:1247
3. Переверзев А.П., Маркина Н.М., Янушевич Ю.Г., Городничева Т.В., Минасян Б.Э., Лукьянов К.А., Гурская Н.Г. (2013). УСИЛЕНИЕ ЭКСПРЕССИИ ХИМЕРНЫХ ГЕНОВ ВКЛЮЧЕНИЕМ В ИХ 3’-НЕТРАНСЛИРУЕМУЮ ОБЛАСТЬ ИНТРОНА 2 ГЕНА БЕТА-ГЛОБИНА ЧЕЛОВЕКА. Биоорг. хим. 40 (3), 293–296 [+]

   Исследована возможность увеличения гетерологичной экспрессии в клетках млекопитающих при включении интронов в 3'нетранслируемую область cоответствующего гена. Для этого в экспресси онный вектор, содержащий ген флуоресцентного белка TagGFP2, вводили интрон 2 гена βглобина человека. Данная последовательность встраивалась после стопкодона TagGFP2 на расстоянии 35 нт. от стопкодона до сайта сплайсинга. Это позволило избежать процесса нонсенсопосредован ной деградации мРНК. Использование второй рамки считывания, кодирующей красный флуорес центный белок Katushka, позволило нормировать уровень трансфекции и экспрессии химерных конструкций. Проточная цитофлуориметрия культуры клеток HEK293T, временно трансфициро ванных генетическими конструкциями, содержащими и не содержащими интрон, позволила вы явить увеличение интенсивности флуоресценции в зеленом канале для конструкции с интроном в 1.8 ± 0.2 раза относительно контрольной безинтронной конструкции. Аналогичное усиление уровня экспрессии в 1.7 ± 0.2 раза при наличии интрона 2 βглобина 3'нетранслируемой области было показа но также для гена, кодирующего дестабилизированный вариант флуоресцентного белка TurboYFP. Эффект увеличения уровня экспрессии химерных конструкций в клетках млекопитающих введени ем интрона в 3'нетранслируемую область целевого гена можно использовать в различных модель ных системах, где введение 5'концевого интрона в последовательность является неприемлeмым.

   ID:1272
4. Gurskaya N.G., Staroverov D.B., Zhang L., Fradkov A.F., Markina N.M., Pereverzev A.P., Lukyanov K.A. (2012). Analysis of alternative splicing of cassette exons at single-cell level using two fluorescent proteins. Nucleic Acids Res. 40 (8), e57 [+]

   Alternative splicing plays a major role in increasing proteome complexity and regulating gene expression. Here, we developed a new fluorescent protein-based approach to quantitatively analyze the alternative splicing of a target cassette exon (skipping or inclusion), which results in an open-reading frame shift. A fragment of a gene of interest is cloned between red and green fluorescent protein (RFP and GFP)-encoding sequences in such a way that translation of the normally spliced full-length transcript results in expression of both RFP and GFP. In contrast, alternative exon skipping results in the synthesis of RFP only. Green and red fluorescence intensities can be used to estimate the proportions of normal and alternative transcripts in each cell. The new method was successfully tested for human PIG3 (p53-inducible gene 3) cassette exon 4. Expected pattern of alternative splicing of PIG3 minigene was observed, including previously characterized effects of UV light irradiation and specific mutations. Interestingly, we observed a broad distribution of normal to alternative transcript ratio in individual cells with at least two distinct populations with ∼45% and >95% alternative transcript. We believe that this method is useful for fluorescence-based quantitative analysis of alternative splicing of target genes in a variety of biological models.

   ID:1271