title: STaQTool: Spot tracking and quantification tool for monitoring splicing of single pre-mRNA molecules in living cells.
abstract: 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.
authors: Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, 1649-028 Lisboa, Portugal. Electronic address: joserino@medicina.ulisboa.pt.
authors: José
authors: J
authors: Rino
authors: Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, 1649-028 Lisboa, Portugal.
authors: Ana C
authors: AC
authors: de Jesus
authors: Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, 1649-028 Lisboa, Portugal.
authors: Maria
authors: M
authors: Carmo-Fonseca
mesh_terms: D036022:Capsid Proteins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D005091:Exons; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000706:statistics & numerical data*; D007438:Introns; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D016366:Open Reading Frames; D012322:RNA Precursors / Q000235:genetics* / Q000378:metabolism; D022821:RNA Splice Sites; D012326:RNA Splicing; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D000072760:Single Molecule Imaging / Q000379:methods / Q000706:statistics & numerical data*; D012984:Software; D000097573:Red Fluorescent Protein
chemical_list: D036022:Capsid Proteins; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D012322:RNA Precursors; D022821:RNA Splice Sites; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D000097573:Red Fluorescent Protein
keywords: Fluorescence microscopy; Fluorescence quantification; Live-cell imaging; RNA splicing; Single-molecule