Pereverzev AP et al. (2015), Method for quantitative analysis of nonsense-me...

XB-ART-50098

Sci Rep 2015 Jan 12;5:7729. doi: 10.1038/srep07729.

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Method for quantitative analysis of nonsense-mediated mRNA decay at the single cell level.

Pereverzev AP , Gurskaya NG , Ermakova GV , Kudryavtseva EI , Markina NM , Kotlobay AA , Lukyanov SA , Zaraisky AG , Lukyanov KA .

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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.

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Species referenced: Xenopus laevis
Genes referenced: smg5 tram1 upf1

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	Figure 1. Scheme of the proposed method of NMD analysis.(a) Main elements in the reporter vectors pNMD+ (left) and pNMD− (right). Brown arrows – CMV promoters. Red arrows – Katushka coding region. Green arrows – TagGFP2 coding region. Grey arrows – human β-globin (HBB) gene fragment. (b) Schematic outline of the reporter function. The pNMD+ vector (left) carries two fluorescent proteins; one (GFP) is encoded by the NMD-targeted transcript, and the other (RFP) serves as an expression efficiency control. The pNMD− control vector (right) encodes both RFP and GFP by NMD-independent transcripts. Comparison of the green-to-red fluorescence ratios between pNMD+ and pNMD− samples allows for the calculation of NMD activity. Brown arrows – promoters. Red, green and grey rectangles – coding regions for RFP, GFP and β-globin, respectively. Blue circles – transcription terminators. Red ″STOP″ signs – stop codons. Red and green cylinders – translated GFP and RFP proteins.
	Figure 2. Fluorescence microscopy of HEK293T cells transiently expressing pNMD+ and pNMD−.Cells were imaged under the same settings. Scale bar: 100 μm.
	Figure 3. Performance of the splicing-dependent NMD reporter in HEK293T cells.(a) Flow cytometry analysis (dot plots in green and red channels) of the cells transiently transfected with pNMD+ (left plot) or pNMD− (right plot). Samples were analyzed using the same flow cytometer settings. To simplify the comparison, the area of pNMD− cells is outlined by a gray dashed line. (b,c) NMD activity determined by fluorescence reporter analysis (b) or quantitative PCR (c) of HEK293T cells transiently transfected with reporter plasmids. Note the strong inhibition of NMD by treatment of the cells with wortmannin (WM), caffeine (Caf), anti-UPF1 shRNA (shUPF1) and cycloheximide (CHX). (c) Assessment of the stability of NMD reporter TagGFP2-encoded mRNA. TagGFP2 mRNA levels were measured by qPCR using HEK293T cells transfected with pNMD− or pNMD+ that were either untreated, treated for 1 h with actinomycin D (AMD) or treated for 1 h with both AMD and CHX. All values were normalized to the untreated pNMD− sample (first column). The mean data of at least 3 independent experiments with the standard deviation are shown in all panels.
	Figure 4. Splicing-dependent NMD reporter in different cell lines.(a–d) Flow cytometry analysis of the cells transiently transfected with pNMD+ (left plots) or pNMD− (right plots). Corresponding pairs of cell samples were analyzed using the same flow cytometer settings. The area of the corresponding NMD− cells is outlined by a gray dashed line on each NMD+ plot. (a) HeLa cells. (b) MEF cells. (c) ES cells. (d) Overgrown HEK293T cells. Note the clear heterogeneity of the NMD activity in these cells. Green and blue dashed lines show the cell populations R1 and R2 with different NMD activities. (e) NMD activity determined by the fluorescence reporter analysis in the designated cell lines. The mean data and standard deviation of at least 3 independent experiments are shown.
	Figure 5. Long 3'UTR-dependent NMD reporter.(a) Main elements in the reporter vectors pSMG5-NMD+ (left) and pTRAM1-NMD− (right). Brown arrows – CMV promoters. Red arrows – Katushka coding region. Green arrows – TagGFP2 coding region. Grey arrows – 3'UTRs of SMG5 and TRAM1. (b,c) Flow cytometry analysis of HeLa (b) and HEK293T (c) cells transiently transfected with pSMG5-NMD+ (left plots) or pTRAM1-NMD− (right plots). Corresponding pairs of cell samples were analyzed using the same flow cytometer settings. The area of the corresponding NMD− cells is outlined by a gray dashed line on the NMD+ plots. (d,e) NMD activity in HeLa or HEK293T cells (untreated or treated with caffeine (Caf), anti-UPF1 shRNA (shUPF1) or cycloheximide (CHX)). Data were obtained by fluorescence reporter analysis (d) or qPCR (e). The mean data and standard deviation of at least 3 independent experiments are shown.
	Figure 6. Splicing-dependent NMD reporter in developing Xenopus embryos.(a) Fluorescence microscopy of representative embryos expressing pNMD+ (upper panels) or pNMD− (bottom panels). Embryos were photographed in white light (upper rows) and the green (middle rows) and red (bottom rows) fluorescence channels. All embryos were imaged under the same settings, except those at stage 45, for which the exposures were decreased to avoid signal saturation. Scale bar: 1 mm. (b) The change of NMD activity during Xenopus development. Data for 7 embryos in 3 independent experiments are shown as a box plot (mean, median, standard deviation, and extrema).

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Bayramov, Novel functions of Noggin proteins: inhibition of Activin/Nodal and Wnt signaling. 2011, Pubmed, Xenbase