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Classical and emerging techniques to identify and quantify localized RNAs

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Abstract In essentially every cell, proteins are asymmetrically distributed according to their function. For many genes, this protein sorting problem is solved by transporting RNA molecules encoding the protein, rather than the protein itself, to the desired subcellular location. The protein is then translated on‐site to immediately produce a correctly localized protein. This strategy is widely used as thousands of RNAs localize to distinct locations across diverse cell types and species. One of the fundamental challenges to study this process is the determination of the subcellular spatial distribution of any given RNA. The number of tools available for the study of RNA localization, from classical and state‐of‐the‐art methods for the visualization of individual RNA molecules within cells to the profiling of localized transcriptomes, is rapidly growing. These include imaging‐based approaches, a variety of biochemical and mechanical fractionation techniques, and proximity‐labeling methods. These procedures allow for both the detailed study of the molecular requirements for the localization of individual RNA molecules and computational studies of RNA transport on a genomic scale. Together, they have the ability to allow insight into the regulatory principles that govern the localization of diverse RNAs. These new techniques provide the framework for integrating our knowledge of the regulation of RNA localization with that of other posttranscriptional processes. This article is categorized under: RNA Export and Localization > RNA Localization RNA Interactions with Proteins and Other Molecules > Protein–RNA Interactions: Functional Implications RNA Methods > RNA Analyses in Cells
The diversity of systems in which the control of RNA localization is critical. (a) In Saccharomyces cerevisiae, ASH1 transcripts are transported to budding daughter cells. This is a key process in mating type switching. (b) In the developing Drosophila embryo, nanos and oskar mRNAs accumulate near the posterior pole of the embryo while bicoid mRNAs accumulate near the anterior end. This arrangement contributes to proper body patterning. (c) In fibroblasts, beta‐actin mRNAs accumulate at the leading edges of projections, contributing to cell motility. (d) In neurons, beta‐actin mRNAs are trafficked to neurites
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DBF‐based proximity labeling approaches. The DBF fluorophore, in the presence of blue light and propargylamine, can catalyze the labeling of nearby RNA molecules with alkyne moieties. These can then be labeled in vitro with any azide‐containing molecule using CuAAC‐mediated “Click” chemistry. In this case, reaction with biotin azide results in the biotinylation of alkyne‐containing RNA molecules, facilitating their purification and analysis
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APEX‐based proximity labeling approaches. The APEX2 enzyme, in the presence of biotin phenol and hydrogen peroxide, can catalyze the biotinylation of nearby RNA molecules. By restricting the expression of APEX2 to subcellular regions of interest, RNA molecules in those regions can be labeled and purified
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Imaging based systems for the analysis of multiple genes. (a) seqFISH uses multiple fluorescent probes and multiple rounds of hybridization. In each hybridization round, the probes that hybridize to a given transcript species are labeled with only one fluorophore. When all of the hybridization rounds are considered, each transcript species will therefore be labeled with different sequence of fluorophores across the rounds. By comparing this sequence to a “lookup table” the identity of the transcripts can be determined. (b) MERFISH uses only a single fluorophore, but in each round of hybridization, only a subset of transcript species will be labeled. When all of the hybridization rounds are considered, each transcript species will have a particular sequence of “on” rounds and “off” rounds (represented as 1 and 0 bits, respectively). By comparing this sequence of bits to a “lookup table,” the identity of the transcript can be determined. The bit sequences within the lookup table are optimized in terms of their similarities to reduce misidentification events
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RNA localization methods for the analysis of single genes. (a) smFISH probes, each with a single fluorophore, are tiled across the transcript of interest. The colocalization of many probes to a single transcript produces a detectable signal. (b) Hairpin arrays, often added to the 3′ UTRs of transcripts, are specifically recognized by viral coat proteins. If these coat proteins are fused to fluorescent proteins, the localization and movement of RNA molecules in living cells can be analyzed. Arrays often contain up to 24 hairpins. (c) RNA aptamers have been designed for small molecules. As with the hairpin arrays, these can be fused to transcripts. Often, the small molecules that are used are cell‐permeable and are not fluorescent until bound to the aptamer, reducing background fluorescence signal. (d) Inactivated CRISPR/Cas proteins can be programmed with guide RNAs that recognize specific transcripts. If the Cas protein is fused to a fluorescent protein, the location of the transcript can be determined
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RNA Methods > RNA Analyses in Cells
RNA Interactions with Proteins and Other Molecules > Protein–RNA Interactions: Functional Implications
RNA Export and Localization > RNA Localization

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