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Following the messenger: Recent innovations in live cell single molecule fluorescence imaging

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Abstract Messenger RNAs (mRNAs) convey genetic information from the DNA genome to proteins and thus lie at the heart of gene expression and regulation of all cellular activities. Live cell single molecule tracking tools enable the investigation of mRNA trafficking, translation and degradation within the complex environment of the cell and in real time. Over the last 5 years, nearly all tools within the mRNA tracking toolbox have been improved to achieve high‐quality multi‐color tracking in live cells. For example, the bacteriophage‐derived MS2‐MCP system has been improved to facilitate cloning and achieve better signal‐to‐noise ratio, while the newer PP7‐PCP system now allows for orthogonal tracking of a second mRNA or mRNA region. The coming of age of epitope‐tagging technologies, such as the SunTag, MoonTag and Frankenbody, enables monitoring the translation of single mRNA molecules. Furthermore, the portfolio of fluorogenic RNA aptamers has been expanded to improve cellular stability and achieve a higher fluorescence “turn‐on” signal upon fluorogen binding. Finally, microinjection‐based tools have been shown to be able to track multiple RNAs with only small fluorescent appendages and to track mRNAs together with their interacting partners. We systematically review and compare the advantages, disadvantages and demonstrated applications in discovering new RNA biology of this refined, expanding toolbox. Finally, we discuss developments expected in the near future based on the limitations of the current methods. This article is categorized under: RNA Export and Localization > RNA Localization RNA Structure and Dynamics > RNA Structure, Dynamics, and Chemistry RNA Interactions with Proteins and Other Molecules > RNA–Protein Complexes
Key steps of the mRNA life cycle can be visualized at the single molecule level. Transcription (1), mRNA processing (2), small RNA processing (3), export (4), RNP import (5), translation (6), regulation by small RNAs (7), mRNA storage (8), and transport (9) all have been examined using the intracellular single molecule visualization techniques reviewed here
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iSHiRLoC presents a set of tools that enables the probing of the intracellular behaviors of mRNPs. Exogenously labeled mRNAs, containing fluorescent nucleotides, are delivered into the cell by microinjection. Co‐injecting labeled mRNAs with a fluorescent injection marker such as high‐molecular weight Dextran allows the injected cellular compartment to be marked (left). Single particle tracking and photobleaching analyses provide a range of physiologically relevant parameters to be extracted from live and fixed‐cell images (right)
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State‐of‐the‐art mRNA and nascent‐peptide visualization strategies. Fluorescently tagged RNA‐binding proteins such as MS2, PP7 and λN bind to specific RNA secondary structures, typically incorporated into the 3′UTR of an mRNA to prevent interference with translation. When placed in the coding sequence (CDS), displacement of these proteins by the translocation of the ribosome can be used to infer protein translation. During translation, the elongating nascent peptide itself can be visualized using nanobodies tagged with fluorescent proteins. Fluorescent RNA aptamers can be used in cis or trans to visualize mRNAs
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RNA Export and Localization > RNA Localization
RNA Interactions with Proteins and Other Molecules > RNA–Protein Complexes
RNA Structure and Dynamics > RNA Structure, Dynamics, and Chemistry

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