RESUMEN
RNA editing results in the site-specific conversion of adenosine to inosine in mRNAs. Genomics has revealed millions of editing sites in metazoans, but examining the spatial aspects of editing in cells has been challenging. A new method, inosineFISH (inoFISH), provides the ability to detect edited and unedited mRNAs within intact cells.
Asunto(s)
Núcleo Celular/genética , Edición de ARN/genética , ARN Mensajero/genética , Animales , Humanos , Hibridación Fluorescente in Situ , Inosina/químicaRESUMEN
The transcriptional kinetics of RNA polymerase II, the enzyme responsible for mRNA transcription in the nucleoplasm, can be modulated by a variety of factors. It is therefore important to establish experimental systems that will enable the readout of transcription kinetics of specific genes as they occur in real time within individual cells. This can be performed by implementing fluorescent tagging of the mRNA under live-cell conditions. This chapter describes how to generate fluorescently tagged genes and mRNA, and how a photobleaching approach can produce information on mRNA transcription kinetics.
Asunto(s)
Recuperación de Fluorescencia tras Fotoblanqueo/métodos , Imagen Molecular/métodos , ARN Polimerasa II/metabolismo , ARN Mensajero/química , Transcripción Genética , Técnicas de Cultivo de Célula/instrumentación , Técnicas de Cultivo de Célula/métodos , Línea Celular , Núcleo Celular/metabolismo , Recuperación de Fluorescencia tras Fotoblanqueo/instrumentación , Genes Reporteros , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Humanos , Cinética , Microscopía Confocal/instrumentación , Microscopía Confocal/métodos , Imagen Molecular/instrumentación , Fotoblanqueo , Plásmidos/genética , ARN Polimerasa II/química , ARN Mensajero/genética , Imagen de Lapso de Tiempo/instrumentación , Imagen de Lapso de Tiempo/métodosRESUMEN
S-adenosylhomocysteine hydrolase (AHCY) is thought to be located at the sites of ongoing AdoMet-dependent methylation, presumably in the cell nucleus. Endogenous AHCY is located both in cytoplasm and the nucleus. Little is known regarding mechanisms that drive its subcellular distribution, and even less is known on how mutations causing AHCY deficiency affect its intracellular dynamics. Using fluorescence microscopy and GFP-tagged AHCY constructs we show significant differences in the intensity ratio between nuclei and cytoplasm for mutant proteins when compared with wild type AHCY. Interestingly, nuclear export of AHCY is not affected by leptomycin B. Systematic deletions showed that AHCY has two regions, located at both sides of the protein, that contribute to its nuclear localization, implying the interaction with various proteins. In order to evaluate protein interactions in vivo we engaged in bimolecular fluorescence complementation (BiFC) based studies. We investigated previously assumed interaction with AHCY-like-1 protein (AHCYL1), a paralog of AHCY. Indeed, significant interaction between both proteins exists. Additionally, silencing AHCYL1 leads to moderate inhibition of nuclear export of endogenous AHCY.
Asunto(s)
Adenosilhomocisteinasa/genética , Adenosilhomocisteinasa/metabolismo , Mapas de Interacción de Proteínas/genética , Transporte Activo de Núcleo Celular/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Citoplasma/genética , Citoplasma/metabolismo , Ácidos Grasos Insaturados/farmacología , Eliminación de Gen , Humanos , Microscopía Fluorescente , Mutación , Unión ProteicaRESUMEN
Discriminating between the mRNA and protein outputs of each of the alleles of an endogenous gene in intact cells, is a difficult task. To examine endogenous transcripts originating from a specific allele, we applied Central Dogma tagging (CD-tagging), which is based on a tag insertion into an endogenous gene by creation of a new exon. Previously, CD-tagging was used to tag endogenous proteins. Here we developed a CD-tagging-MS2 approach in which two tags were inserted in tandem; a fluorescent protein tag in conjunction with the mRNA MS2 tag used for tagging mRNAs in cells. A cell clone library of CD-tagged-MS2 genes was generated, and protein and mRNA distributions were examined and characterized in single cells. Taking advantage of having one allele tagged, we demonstrate how the transcriptional activity of all alleles, tagged and untagged, can be identified using single molecule RNA fluorescence in situ hybridization (smFISH). Allele-specific mRNA expression and localization were quantified under normal and stress conditions. The latter generate cytoplasmic stress granules (SGs) that can store mRNAs, and the distribution of the mRNAs within and outside of the SGs was measured. Altogether, CD-tagging-MS2 is a robust and inexpensive approach for direct simultaneous detection of an endogenous mRNA and its translated protein product in the same cell.