RESUMO
Nonsense-mediated mRNA decay (NMD) is a mechanism of mRNA surveillance ubiquitous among eukaryotes. Importantly, NMD not only removes aberrant transcripts with premature stop codons, but also regulates expression of many normal genes. A recently introduced dual-color fluorescent protein-based reporter enables analysis of NMD activity in live cells. In this chapter we describe the method to generate stable transgenic cell lines expressing the splicing-dependent NMD reporter using consecutive steps of lentivirus transduction and Tol2 transposition.
Assuntos
Linhagem Celular/metabolismo , Genes Reporter/genética , Engenharia Genética/métodos , Proteínas de Fluorescência Verde/metabolismo , Degradação do RNAm Mediada por Códon sem Sentido/genética , Animais , Separação Celular/instrumentação , Separação Celular/métodos , Citometria de Fluxo/instrumentação , Citometria de Fluxo/métodos , Proteínas de Fluorescência Verde/genética , Humanos , Camundongos , Microscopia de Fluorescência/métodos , Splicing de RNA/genética , RNA Mensageiro/metabolismo , Transfecção/métodos , Transgenes/genéticaRESUMO
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.
Assuntos
Degradação do RNAm Mediada por Códon sem Sentido/genética , Análise de Célula Única/métodos , Regiões 3' não Traduzidas/genética , Animais , Embrião não Mamífero/metabolismo , Citometria de Fluxo , Genes Reporter , Vetores Genéticos/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Células HeLa , Humanos , Camundongos , Microscopia de Fluorescência , Splicing de RNA/genética , Xenopus laevisRESUMO
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 â¼45% 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.
