Transcription is a highly dynamic process, which, for many genes, occurs in stochastic bursts. Studying what regulates these stochastic bursts requires visualization and quantification of transcription dynamics in single living cells. Such measurements of bursting can be accomplished by labeling nascent transcripts of single genes fluorescently with the MS2 and PP7 RNA labeling techniques. Live-cell single-molecule microscopy of transcription in real time allows for the extraction of transcriptional bursting kinetics inside single cells. This chapter describes how to set up the MS2 or PP7 RNA labeling system of endogenous genes in both budding yeast (Saccharomyces cerevisiae) and mammalian cells (mouse embryonic stem cells). We include how to genetically engineer the cells with the MS2 and PP7 system, describe how to perform the live-microscopy experiments and discuss how to extract transcriptional bursting parameters of the genes of interest.
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- The study presents new variants of MS2 and PP7 proteins called dMCP and dPCP, designed to improve live-cell RNA imaging by reducing background noise from unbound probes.
- These variants are engineered to degrade quickly unless they are bound to specific RNA targets, enhancing the sensitivity of RNA visualization.
- The research also introduces dual-color imaging capabilities, allowing for simultaneous tracking of different RNA species in the same cell without interference, broadening the potential applications for RNA imaging in research and biotechnology.
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Development
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Faculty of Biology, Medicine and Health, The University of Manchester, Manchester M13 9PT, UK.
Live imaging of transcription in the Drosophila embryo using the MS2 or PP7 systems is transforming our understanding of transcriptional regulation. However, insertion of MS2/PP7 stem-loops into endogenous genes requires laborious CRISPR genome editing. Here, we exploit the previously described Minos-mediated integration cassette (MiMIC) transposon system in Drosophila to establish a method for simply and rapidly inserting MS2/PP7 cassettes into any of the thousands of genes carrying a MiMIC insertion.
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STAR Protoc
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Laboratory of Pollen Biology, Institute of Experimental Botany of the Czech Academy of Sciences, Rozvojová 263, 165 00 Prague, Czech Republic. Electronic address:
Here, we present a protocol for labeling and live visualization of RNA-protein complexes in the form of ribonucleoprotein particles (RNPs) in tobacco pollen tubes. We describe steps for constructing RNA-pp7/MS2 tag and biolistic gene-gun-mediated pollen transformation. We then provide detailed procedures for RNA labeling using PP7 aptamer nascent RNA tagging and a fluorescently labeled Pseudomonas aeruginosa PP7 bacteriophage coat protein (PCP) reporter that binds to PP7 RNA stem loops.
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Department of Chemical and Biomolecular Engineering, University of Pennsylvania, Philadelphia, PA, USA.
Transcription in developing metazoans is inherently stochastic, involving transient and dynamic interactions among transcriptional machinery. A fundamental challenge with traditional techniques, including fixed-tissue protein and RNA staining, is the lack of temporal resolution. Quantifying kinetic changes in transcription can elucidate underlying mechanisms of interaction among regulatory modules.
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