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WIREs Dev Biol
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The many functions of mRNA localization during normal development and disease: from pillar to post

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Abstract The regulated intracellular trafficking and localized translation of mRNA molecules represents an important and prevalent mechanism of gene regulation. This process plays a key role in modulating asymmetric protein distribution linked to a wide variety of biological processes in different organisms and cell types. In this review, we begin by discussing the diverse biological functions, advantages, and mechanisms of mRNA localization that have been characterized to date. We then review recent technological innovations in RNA imaging and functional genomics methods that will undoubtedly provide powerful new strategies for the elucidation of mRNA trafficking pathways. Finally, we discuss several examples linking human disease pathogenesis to defects in transcript localization, which further underlines the critical importance of this gene regulatory mechanism. WIREs Dev Biol 2013, 2:781–796. doi: 10.1002/wdev.113 This article is categorized under: Gene Expression and Transcriptional Hierarchies > Regulatory Mechanisms Gene Expression and Transcriptional Hierarchies > Regulatory RNA Technologies > Analysis of Cell, Tissue, and Animal Phenotypes Technologies > Analysis of the Transcriptome

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Diverse examples of localized mRNAs. Transcript distribution patterns observed in different model organisms and cell types reveal the diverse biological functions of localized mRNAs. (a) In stage 10 Drosophila oocytes, the localization of maternal bicoid (red), oskar (green), and gurken (violet) mRNAs to the anterior, posterior, and anterodorsal corner of the oocyte, respectively, is essential for anteroposterior and dorsoventral patterning of the embryo. (b) Vegetal pole targeting of Vg1 and VegT mRNAs is important for endoderm and mesoderm germ layer specification in Xenopus oocytes. (c) Migrating cells localize β‐actin mRNA to lamellipodia, where its targeted translation supports the assembly of actin filaments required for cell motility. (d) The dendritic localization of transcripts such as CamKIIα in mature mammalian neurons is important for synaptic plasticity associated with learning and memory. (e) In the yeast Saccharomyces cerevisiae, Ash1 mRNA is segregated into the daughter cell during the budding process where it acts to differentially regulate mother–daughter cell fates. (f) In the pumpkin Cucurbita maxima, ribonucleoprotein complexes formed by the RBP50 protein and mRNAs such as GAIP undergo long‐distance intercellular trafficking within the phloem stream of the plant.

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Cellular fractionation coupled with high‐throughput gene expression profiling is a powerful approach for identifying cohorts of localized RNAs. Various cell fractionation strategies can be used to isolate mRNAs from specific subcellular compartments. (a) This includes the use of detergents or ultracentrifugation methods to fractionate cytosolic RNAs (purple) away from transcripts enriched within specific organelles, such as mitochondria and the endoplasmic reticulum. (b and c) Alternatively, mechanical separation (shown with large dashed lines) of cell compartments, such as lamellopodia from migratory cells (b, red region) or axonal growth cones (c, green shading). The large upward arrows in these examples (a–c) indicate the extracted organelles or cell compartments. (d) RNA extracted from specific subcellular fractions can then be identified through microarray or RNA deep sequencing analysis (e.g., Illumina® HiSeq 2000) and subsequent gene expression profiling to characterize the enriched RNA populations.

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Large‐scale in situ hybridization projects have identified new varieties of localized RNAs. The establishment of comprehensively annotated cDNA libraries for a variety of model organisms has enabled the implementation of several large‐scale in situ hybridization (ISH) projects to assess the expression patterns and subcellular localization properties of broad collections of mRNAs. The cDNAs are employed to systematically generate labeled RNA probes for high‐throughput ISH analyses in different model organisms and cell types. Examples of online ISH databases are shown in which the developmental profiles of gene expression and RNA localization have been curated in zebrafish (e.g., ZFIN), mouse brain (e.g., Allen Brain Atlas), and fruit fly embryos (e.g., Fly‐FISH, BDGP in situ homepage).

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Genetic tagging strategies to visualize RNAs in live cells. Several approaches have been developed over the years to visualize RNAs in living cells and organisms. These frequently involve the insertion of genetic tags in the RNA of interest that serve as recognition sites for fluorescently labeled RNA‐binding proteins (RBPs) or fluorogenic small molecules. (a) To date, the most widely used approach has been the MS2 detection system, involving the addition of multimerized MS2 RNA hairpins within an RNA, which is co‐expressed with an MS2 coat protein (MCP)‐GFP fusion. MCP‐GFP dimmers bind to individual MS2 RNA hairpins, thus allowing the visualization of RNA localization properties by fluorescence microscopy. The principal limitation of this approach is the high level of fluorescence background due to unbound fusion protein, which can limit the capacity to clearly visualize RNA movements. (b) An alternative strategy, which can significantly reduce the background signal due to unbound fluorescent proteins, is the use of biomolecular fluorescence complementation. In this approach, the N‐ and C‐terminal fragments of a fluorescent protein (e.g., GFP) are expressed as fusions to different RNA‐binding domains that recognize distinct genetically encoded tags in the RNA of interest. Co‐recruitment of both fusion protein species to the same RNA molecule, through binding to their specific recognition sites, allows reformation of a functional fluorescent protein and visualization of the tagged RNA. (c) More recently, a novel RNA imaging approach was developed through the identification of an RNA aptamer, designated Spinach, that interacts with fluorogenic small molecules (e.g., DFHBI) leading to the emission of bright fluorescent signals.

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General model depicting different mechanisms and molecular functions of mRNA localization. (a) Examples are shown of nuclear mRNAs containing distinct zipcode elements that are recognized by specific RNA‐binding proteins (RBPs), either in the nucleus or in the cytoplasm following mRNA nuclear export, which modulate their trafficking to different regions of the cell. Once transport ribonucleoproteins are formed, they may (b) undergo general diffusion followed by their entrapment in a specific region of the cell; (c) associate with motor proteins that mediate their directional transport on cytoskeletal networks; or (d) become localized through degradation protection. (e) In addition to undergoing targeted translation, localized mRNAs may also carry out noncoding functions, for example, by exerting catalytic or scaffolding activities. (f) In some cases, proteins translated from localized mRNAs can undergo retrograde transport, for instance, to relay signals from the cell periphery back to the nucleus in response to extracellular cues.

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