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Methods for spatial and temporal imaging of the different steps involved in RNA processing at single‐molecule resolution

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Abstract RNA plays a quintessential role as a messenger of information from genotype (DNA) to phenotype (proteins), as well as acts as a regulatory molecule (noncoding RNAs). All steps in the journey of RNA from synthesis (transcription), splicing, transport, localization, translation, to its eventual degradation, comprise important steps in gene expression, thereby controlling the fate of the cell. This lifecycle refers to the majority of RNAs (primarily mRNAs), but not other RNAs such as tRNAs. Imaging these processes in fixed cells and in live cells has been an important tool in developing an understanding of the regulatory steps in RNAs journey. Single‐cell and single‐molecule imaging techniques enable a much deeper understanding of cellular biology, which is not possible with bulk studies involving RNA isolated from a large pool of cells. Classic techniques, such as fluorescence in situ hybridization (FISH), as well as more recent aptamer‐based approaches, have provided detailed insights into RNA localization, and have helped to predict the functions carried out by many RNA species. However, there are still certain processing steps that await high‐resolution imaging, which is an exciting and upcoming area of research. In this review, we will discuss the methods that have revolutionized single‐molecule resolution imaging in general, the steps of RNA processing in which these methods have been used, and new emerging technologies. This article is categorized under: RNA Export and Localization > RNA Localization RNA Methods > RNA Analyses in Cells RNA Interactions with Proteins and Other Molecules > Small Molecule‐RNA Interactions
Single molecule RNA imaging in fixed cells. Methods for imaging RNA target directly with fluorophores in fixed cells using hybridization techniques. (a) Stellaris probes is a commercial name for single‐molecule FISH probes. The cells are fixed, permeabilized, and then hybridized with a set of approximately 50 probes, each 18–20 nucleotides long. Each probe labeled with a single fluorophore binds to a different position along the length of the target RNA, thereby producing a diffraction‐limited spot in the microscope image. The nonspecific binding of a few probes does not yield any discrete signals. (b) Representative image obtained with Stellaris probes, in which merged z‐stacks of HS5 cells were hybridized with probes labeled with Texas red. Each spot represents a single RNA molecule. The scale bar is 5 μm long (image from the Batish Laboratory). (c) RNAscope: sequential binding of z‐probes, pre‐amplifiers, and amplifiers, with washes after each binding step, creates sites for the binding of multiple signal‐generating probes, leading to either fluorogenic or chromogenic visible spots for each target RNA molecule
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CRISPR–Cas9‐based RNA tagging for single‐molecule RNA imaging. A modified Cas9 protein fused with GFP binds to a target RNA with the help of an sgRNA and a PAM‐mer sequence. This leads to the generation of a signal at the site of sgRNA binding, and the RNA becomes visible under a fluorescence microscope
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Genetically encoded sensors for single‐molecule RNA imaging in living cells with aptamers. RNA aptamers bind a nonfluorescent dye with high affinity, restricting its motion, thereby rendering the dye fluorescent. (a) An array of aptamer molecules is cloned into the end of the target RNA and expressed in cells. (b) The cells are grown in media containing the dye, which is nonfluorescent when free within the cell. (c) The binding of the dye to the aptamer sequence leads to the correct folding of the RNA aptamer, resulting in a bright fluorescent signal, which is visible when viewed with a fluorescence microscope
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Single‐molecule RNA imaging by the MS2 system in vivo. (a) This RNA–protein system relies on engineered RNAs that contain stem‐loops (for example MS2 stem‐loops) that are recognized by specific RNA binding proteins (for example MS2 coat protein) that are tagged with fluorescent reporter proteins. An array of RNA stem‐loops are cloned into the 3′ untranslated region (UTR) of the target RNA and expressed in the cells. (b) A fluorescent reporter protein (e.g., green fluorescent protein) is cloned in frame with the stem‐loop binding protein and expressed in cells. (c) As the cells express the engineered RNA, the tagged proteins bind to the engineered target RNAs rendering the RNA visible when viewed with a fluorescence microscope
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Single‐molecule RNA imaging with molecular beacons. Molecular beacons (MB) produce fluorescence only when bound to specific target molecules that are recognized by the loop region in these probes. (a) Although molecular beacons can be designed to bind to endogenous target RNAs, usually an array of molecular beacon binding sites are cloned at the end of the target RNA, and this construct is expressed in cells. (b) Molecular beacons can be introduced into cells by microinjection. These probes have a tendency to accumulate in the nucleus. (c) As the engineered target RNA is expressed, the molecular beacon loop region binds to the complementary target regions in the engineered RNA, resulting in a fluorescence signal that renders the RNA visible when viewed with a fluorescence microscope
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Single‐molecule imaging of RNA–protein complexes in vitro. Co‐localization single‐molecule spectroscopy (CoSMoS) uses two differently, fluorescently labeled parts: the RNA of interest and the protein of interest. The RNA of interest is conjugated to a fluorophore and immobilized on a glass slide. This immobilized RNA is then treated with a purified, fluorescently labeled protein. Visualization of the slide under a microscope reveals single spots indicative of the presence of the RNA or the protein, and co‐localized spots represent protein–RNA complexes
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RNA Interactions with Proteins and Other Molecules > Small Molecule–RNA Interactions
RNA Methods > RNA Analyses in Cells
RNA Export and Localization > RNA Localization

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