RESUMO
Single mRNA molecules can be imaged in living cells by a method that consists in genetically inserting binding sites for a bacteriophage protein in the gene of interest. The resulting reporter transgene is then integrated in the genome of cells that express the phage protein fused to a fluorescent tag. Upon transcription, binding of the fluorescent protein to its target sequence makes the RNA visible. With this approach it is possible to track, in real time, the life cycle of a precursor mRNA at the site of transcription in the nucleus and transport of mature mRNA to the cytoplasm. In order to measure the fluorescence associated with individual RNA molecules over time, we developed a semi-automated quantitative image analysis tool termed STaQTool. We describe in detail the implementation and application of the STaQTool software package, which is a generic tool able to process large 4D datasets allowing quantitative studies of different steps in gene expression.
Assuntos
Processamento de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência/métodos , Imagem Molecular/métodos , RNA Mensageiro/metabolismo , Imagem Individual de Molécula/métodos , Software , Splicing de RNA , RNA Mensageiro/genética , Interface Usuário-Computador , NavegadorRESUMO
The vast majority of human protein-coding genes contain up to 90% of non-coding sequence in the form of introns that must be removed from the primary transcripts or pre-mRNAs. Diverse forms of mRNAs encoded from a single gene are created by the differential use of splice sites and alternative splicing is rapidly evolving. Although the kinetic properties of splicing are thought to be critical for proofreading and regulatory mechanisms, tools for making direct experimental measurements of splicing rates are still limited. We recently developed a strategy that permits real-time imaging of fluorescent-labelled introns in single pre-mRNA molecules. Here we describe the software tool that we created for automatic tracking and quantification of intronic fluorescence at the site of transcription in live human cells.
Assuntos
Processamento de Imagem Assistida por Computador/estatística & dados numéricos , Precursores de RNA/genética , Splicing de RNA , RNA Mensageiro/genética , Imagem Individual de Molécula/estatística & dados numéricos , Software , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Linhagem Celular , Éxons , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Íntrons , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência , Fases de Leitura Aberta , Precursores de RNA/metabolismo , Sítios de Splice de RNA , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Imagem Individual de Molécula/métodos , Proteína Vermelha FluorescenteRESUMO
Microscopy protocols that allow live-cell imaging of molecules and subcellular components tagged with fluorescent conjugates are indispensable in modern biological research. A breakthrough was recently introduced by the development of genetically encoded fluorescent tags that combined with fluorescence-based microscopic approaches of increasingly higher spatial and temporal resolution made it possible to detect single protein and nucleic acid molecules inside living cells. Here, we describe an approach to visualize single nascent pre-mRNA molecules and to measure in real time the dynamics of intron synthesis and excision.
Assuntos
Rastreamento de Células/métodos , Microscopia de Fluorescência/métodos , Precursores de RNA/genética , Splicing de RNA/genética , Proteínas de Fluorescência Verde , Células HeLa , HumanosRESUMO
Expression of genetic information in eukaryotes involves a series of interconnected processes that ultimately determine the quality and amount of proteins in the cell. Many individual steps in gene expression are kinetically coupled, but tools are lacking to determine how temporal relationships between chemical reactions contribute to the output of the final gene product. Here, we describe a strategy that permits direct measurements of intron dynamics in single pre-mRNA molecules in live cells. This approach reveals that splicing can occur much faster than previously proposed and opens new avenues for studying how kinetic mechanisms impact on RNA biogenesis.
