Biological functions, regulatory mechanisms, and disease relevance of RNA localization pathways

Transcription

1 REVIEW ARTICLE Biological functions, regulatory mechanisms, and disease relevance of RNA localization pathways Samantha Bovaird 1,2,*, Dhara Patel 1,3,*, Juan-Carlos Alberto Padilla 1,2,* and Eric Lecuyer 1,2,3,4 1 Institut de recherches cliniques de Montreal (IRCM), QC, Canada 2 Division of Experimental Medicine, Faculty of Medicine, McGill University, Montreal, QC, Canada 3 Molecular Biology Program, Faculty of Medicine, Universite de Montreal, QC, Canada 4 Department of Biochemistry and Molecular Medicine, Universite de Montreal, QC, Canada Correspondence E. Lecuyer, Institut de recherches cliniques de Montreal (IRCM), 110 Avenue des Pins Ouest, Montreal, QC, Canada Fax: Tel: eric.lecuyer@ircm.qc.ca *Samantha Bovaird, Dhara Patel and Juan- Carlos Alberto Padilla contributed equally to this article (Received 2 July 2018, revised 6 August 2018, accepted 17 August 2018, available online 5 September 2018) The asymmetric subcellular distribution of RNA molecules from their sites of transcription to specific compartments of the cell is an important aspect of post-transcriptional gene regulation. This involves the interplay of intrinsic cis-regulatory elements within the RNA molecules with trans-acting RNAbinding proteins and associated factors. Together, these interactions dictate the intracellular localization route of RNAs, whose downstream impacts have wide-ranging implications in cellular physiology. In this review, we examine the mechanisms underlying RNA localization and discuss their biological significance. We also review the growing body of evidence pointing to aberrant RNA localization pathways in the development and progression of diseases. Keywords: Cis-regulatory elements; RNA disease; RNA localization; RNA-binding proteins doi: / Edited by Wilhelm Just The asymmetric organization of cells is a pervasive feature that ensures the homeostasis of prokaryotic and eukaryotic organisms. This relies on the capacity of cells to organize their contents, including lipids, nucleic acids, and proteins, into specific subcellular structures and organelles. While much of this organization has been attributed to subcellular protein transport [1], a growing body of work indicates that the regulated localization of RNA molecules is also a key process observed across evolutionary timescales [2 6]. This form of post-transcriptional regulation where coding and noncoding RNA molecules actively associate with trans-acting partners, such as RNA-binding proteins (RBPs), serves to dictate both the function and intra- or extracellular destiny of the RNA. During RNA localization, cis-regulatory elements found within the RNA molecule, whether coding or noncoding, act as recognition sites for the recruitment of trans-acting RBPs, which dictate the intra-/extra-cellular fate of the RNA and, ultimately, its functional state. Over the years, the wide-ranging implications of RNA localization on cellular physiology have become apparent, affecting many aspects of cell organization and function. Indeed, RNA localization pathways have been linked to numerous important processes, including developmental patterning, cell polarity, spindle assembly, formation of nonmembranous compartments, localized translation, and cell motility [2 6]. Abbreviations ALS, Amyotrophic lateral sclerosis; ASOs, antisense oligonucleotides; DM, Myotonic Dystrophy; DPR, dipeptide repeat; EGFR, epidermal growth factor receptor; EV, extracellular vesicle; IDR, intrinsically disordered regions; MBNL, Muscleblind; MS, mass spectrometry; RAN, repeat-associated non-atg-dependent; RBDs, RNA binding domains; RBPs, RNA-binding proteins; RNP, ribonucleoprotein; SGs, stress granules; SNRs, short nucleotide repeat expansions FEBS Letters 592 (2018) ª 2018 Federation of European Biochemical Societies

2 S. Bovaird et al. RNA localization: functions and disease links Recently, alterations to normal RNA localization pathways have also been increasingly implicated in disease etiology. Here, we discuss some of the historical milestones in the field of RNA localization, starting with some of the early pioneer studies to the more recent highthroughput and genome-wide approaches. We review some of the known biological functions fulfilled by localized RNAs and associated machineries, as well as the prevalence of these biological processes in gene regulation. Next, we examine some of the established molecular mechanisms and postulated advantages of this regulatory system, including recent findings on the association of RNA localization machineries and the formation of membraneless structures. Finally, we explore the involvement of abnormal RNA localization pathways in the development and progression of several diseases, thereby underlining the importance of understanding these important mechanisms. Biological functions and prevalence of RNA localization The earliest reports of subcellular localization of specific RNA molecules date back to the mid-1980s/early- 1990s, where examples of localized transcripts were characterized across a number of model organisms and cell types [7 16] (see examples in Fig. 1). This included delimitation of actin messenger RNA (mrna) to the myoplasm, ectoplasm, and endoplasm in ascidian eggs and embryos [7]; the localization of b-actin mrna to the cell extremities of chicken myoblasts and fibroblasts [8]; and the enrichment of transcripts along the animal-vegetal or anterior-posterior axes of developing eggs in Xenopus and Drosophila [9 16]. These examples established the important roles played by localized RNAs in modulating cell migration, cell fate determination, and embryonic axis specification [3 5,17,18]. Since these early days, RNA localization has been observed in a diverse range of species, from bacteria to humans [3 5,18]. Below, we discuss some of the established functions of localized mrnas and the emergence of transcriptomic studies, indicating that RNA localization is a highly prevalent process. Molecular functions of RNA localization The subcellular trafficking of RNA molecules can have diverse functional roles, whether one considers mrnas or the various biotypes of noncoding RNAs encoded in genomes. Firstly, as different steps of RNA processing (e.g., splicing, editing, degradation, translation) are thought to take place in different subregions of the cell, each implicating specific ribonucleoprotein (RNP) machineries, the proper targeting of regulatory RNAs and their targets is likely to be critical for accurate post-transcriptional gene regulation, both under steady-state and stress conditions. For instance, in mammalian cell nuclei, splicing and maturation of premrnas is thought to be carried out in specific subnuclear bodies designated as nuclear speckles, which are enriched in splicing regulators and RNA nuclear export factors [19]. By contrast, in rat hippocampal neurons, the localization of microrna mir-134 to the synaptodendritic compartment was shown to prevent the translation of Limk mrna, consequently controlling the dendritic spine volume [20,21]. In response to various stresses, cellular mrnas are translationally silenced as they undergo recruitment into cytoplasmic membrane-less bodies termed stress granules (SGs), where they are transiently stored until the stress dissipates, and allowing re-entry of mrnas into an active translation state [22 25]. These wide-ranging examples, though seemingly disconnected, highlight the importance of organizing post-transcriptional regulatory machineries in subcellular space to achieve desired regulatory outcomes in different cellular and environmental contexts. In the case of mrnas, the functional benefits of localization are usually viewed through the lens of spatially regulated translation control. Indeed, the process of localized translation can offer an exquisite strategy to enhance the precision of delivery of proteins to locations where they are needed in the cell, while minimizing the risk of mis-targeting. Considering that a single mrna molecule can be translated into hundreds of protein products, localized translation would seem energetically cost-effective, since many copies of the encoded protein can be produced in the desired compartment. Recent estimates of ribosomal occupancy based on live cell imaging studies indicate that individual mrnas are typically coated with ribosomes and can undergo rapid transitions in translational activity in specific subcellular compartments and in response to environmental stimuli [26,27]. These observations were made using the SunTAG system, a technique that relies on the introduction of genetically encoded multimerized SunTAG sequences upstream of an mrna of interest. When this construct is translated, a co-expressed GFP-fused single chain antibody can bind to the SunTAG peptides as they are translated by the ribosome, without requiring a fully matured protein product, thus allowing one to visualize the sites of de novo protein synthesis with high precision [26,27]. Localized mrna translation may also serve to prevent the translated proteins from accessing FEBS Letters 592 (2018) ª 2018 Federation of European Biochemical Societies 2949

3 RNA localization: functions and disease links S. Bovaird et al. areas of the cell where it may go to waste or have deleterious consequences. For example, mis-localization of mrnas encoding embryonic polarity regulators can profoundly disrupt developmental patterning events [28]. Similarly, targeting myelin basic protein mrna to the distal ends of oligodendrocytes is important to prevent unwanted membrane fusion events [29,30]. The localized translation of mrnas encoding functionally-associated proteins may also yield more efficient protein complex assembly [31 33]. One of the earliest examples of this occurrence was depicted in the amoeba, where it was found that the mrnas of the components that make up the basal body and flagella were co-localized to the cell periphery [31]. In another example, the individual mrnas of the Arp2/3 multiprotein complex exhibited similar localization patterns at the cell protrusions of fibroblasts [34]. Regulated trafficking of mrnas may also serve purposes other than localized translation. For example, VegT mrna has been shown to play coding-independent roles in the anchoring of the cytokeratin cytoskeleton in Xenopus oocytes [35]. In mouse hepatic cells, a distinct population of cat2 RNA is retained in nuclei until stress conditions lead to its cleavage to produce a protein-coding mrna molecule that can be translated [36]. In another instance, a specific collection of early zygotic mrnas exhibited nuclear retention when Drosophila embryos were exposed to DNA damaging agents, thus preventing their translation [37]. This process is part of a quality control checkpoint that serves to eliminate damaged nuclei from the embryonic cell progenitor pool. These various examples serve to illustrate some of the functional benefits of RNA trafficking pathways. General prevalence of RNA localization With the accumulation of comprehensive studies using high-throughput imaging or subcellular transcriptomics profiling to assess RNA distribution features on a transcriptome-wide scale, RNA localization has emerged as a highly prevalent regulatory step in the control of gene expression [4,38]. For example, large-scale fluorescent in situ hybridization studies on Drosophila oocytes and embryos showed that a large proportion of mrnas and long noncoding RNAs (lncrna) exhibit precise localization to specific organelles and subcellular structures (e.g., plasma membrane, mitotic apparatus, cell junctions, subnuclear domains), emphasizing the high prevalence of this mechanism [39 41]. Moreover, a large number of studies have employed biochemical purification and RNA expression profiling strategies to characterize organelle-enriched RNA populations, including both coding and noncoding RNA species [39,42 58]. In addition to documenting localized transcriptomes, some studies have also begun using such comprehensive approaches to assess how RNA cytotopic distribution patterns are altered under different physiological states or in response to genetic perturbations [43,49]. For instance, in a recent study by Moor et al., the importance of polarized mrna distribution along the apical and basolateral axis of intestinal epithelium of the mouse was highlighted [43]. This work showed that hundreds of mrnas are transported from the basal lamina to the ribosome-enriched apical cytoplasm of intestinal epithelial cells when fasted mice were refed, thereby revealing the dynamic nature of global cellular mrna arrangements [43]. Several groups have also simultaneously profiled RNA and protein expression across subcellular compartments in order to assess the general coherence in expression signatures [44,59,60]. For instance, Zappulo et al. recently used RNA-seq, ribosome profiling and mass spectrometry (MS) to define the local transcriptomes, translatomes and proteomes of cell bodies and neurites of induced neurons derived from mouse embryonic stem cells [44]. Remarkably, they found that approximately 50% of neurite-resident proteins were targeted via local mrna translation, arguing that mrna localization is a major determinant of protein targeting to this compartment. Using a combined approach of RNA-seq and MS profiling of human and Drosophila cells fractionated into distinct biochemically defined compartments (i.e., nuclear, insoluble, endomembrane, and cytosol), Benoit Bouvrette et al. found that the majority of mrnas and noncoding RNA biotypes are asymmetrically distributed in patterns that are generally conserved evolutionarily [60]. Comparative analysis of mrna and protein distribution profiles revealed a general lack of correlation, a feature that was strongly influenced by specific families of transcripts exhibiting strong asymmetric distribution with their encoded protein products. For instance, mrnas encoding DNA-binding transcription factors and chromatin components/regulators need to undergo translation in the cytoplasm, while their protein products are typically strongly enriched in the nucleus. However, when this fractionation data was dissected further and segmented to interrogate mrnas encoding components of known protein modules, functionally-related transcripts tended to exhibit similar fractionation profiles, while the degree of co-fraction with their encoded proteins varied depending on protein complexes under study [60]. For example, mrnas for components of the Arp2-3 complex, known to undergo transport to 2950 FEBS Letters 592 (2018) ª 2018 Federation of European Biochemical Societies

4 S. Bovaird et al. RNA localization: functions and disease links cell protrusions [34], showed close co-fractionation with the their protein products. This type of analysis can thus be useful in identifying molecular machines that are likely enriched at specific subcellular compartments through localized translation. Finally, similar to mrna profiling efforts, these types of subcellular transcriptomics studies have also revealed the extensive asymmetry in distribution patterns displayed by various families of noncoding RNAs, including lncrnas, mirnas, and circular RNAs [44,60 63]. Collectively, these approaches highlight that high prevalence and diversity of RNA localization events. Mechanisms of RNA localization To grapple with the multifaceted modes of RNA localization and their linkage to cellular function, we must first address the principles governing the interaction between RNA transcripts and their trans-acting-binding partners. Like most steps of gene regulation, localization control typically relies on the presence of specific cis-regulatory elements within the RNA molecule, often designated as RNA zipcodes, which are themselves recognized by trans-acting machineries. These trans-regulators have classically been defined as RBPs, but may also include regulatory RNA molecules. The assembly of RNP complexes, formed by a localized RNA and its associated trans-regulators, is thought to be a dynamic process involving distinct regulatory steps carried out in the nucleus and cytoplasm. Indeed, once exported into the cytoplasm, RNPs may be remodeled through the release and acquisition of new components, and may further assemble into higher order transport granules through specific protein-protein and protein RNA interactions [64 68]. For instance, oskar mrna in the early Drosophila embryo oligomerizes via its 3 0 UTR, mediated by polypyrimidine tract-binding protein [69,70]. This permits both stepwise association of other factors to the transport granule and effectively represses translation of oskar mrna during transport toward the posterior pole [69,70]. Thus, cells are thought to harbor different varieties of transport granules packed with functionally relevant RBPs and accessory factors that can jointly modulate different aspects of RNA regulation, including localization, stability, and translational status [4]. Three predominant mechanisms of RNA localization have been characterized, each reliant on cis-acting RNA sequences and the trans-acting elements [71]. The first, and perhaps most pervasive mode of RNA trafficking, involves directed cytoskeletal transport, in which transport RNPs associate with the cytoskeleton through interaction with specific motor proteins, thus enabling direct transport toward specific subcellular regions [68]. For instance, the Vg1 mrna, required in the vegetal pole specification in Xenopus embryos, is bound at the vegetal localization element (VLE) in its 3 0 UTR by the Vera RBP, which associates with microtubules [72,73]. Similarly, the mrnps involving ZBP1 and b-actin mrna, are directed along microtubules in axonal projections by the Kinesin Family 11 (KIF11) motor protein or along lamellipodia in fibroblasts [74 76]. Cytoskeletal transport may also be bidirectional, as uncovered for KIF5-mediated transport of CaMKIIalpha and Arc mrna transport granules, which move in a bidirectional manner along dendrites [67]. A second mode of RNA localization involves localized protection from degradation. In early Drosophila embryos, Hsp83 transcripts are localized to the posterior pole through this mechanism [77]. In this example, the Smaug protein, localized throughout the embryo in a decreasing anterior-to-posterior concentration gradient, binds to the 3 0 UTR of Hsp83 and induces degradation of the transcript via recruitment of CCR4-NOT deadenylase to Hsp83 [78]. The nanos mrna, which is also localized to the posterior pole in the Drosophila embryo, undergoes a similar mechanism: Smaug, enriched in the anterior germplasm, binds to the nanos 3 0 UTR to induce its degradation. [79,80]. This causes a local increase of nanos concentration of 100-fold in the posterior pole [79]. The third broad mode of subcellular targeting, termed diffusion and entrapment, relies on anchoring of the transcript to a specific compartment. This mode is also exhibited by nanos mrna, wherein cytoplasmic flow causes diffusion of nanos to actin-based anchors located specifically at the posterior pole [81,82]. This mode is less efficient in the case of nanos, yet the combination of both localized protection from degradation and diffusion and entrapment in the germplasm permit its tightly regulated spatial distribution [81]. Evidently, the broad modes of RNA localization provide a basis for complex targeting mechanisms and may act in tandem to precisely localize a given RNA transcript. Below, we delve more deeply into the features of cisand trans-regulatory elements (Fig. 2A,B), while also exploring the propensity of these interactions to occur in the context of membrane-less granules (Fig. 2C). Cis-acting RNA localization regulatory elements Cis-acting RNA zipcode sequences serve as a vital physical foundation of molecular interactions influencing subcellular targeting. Classical methods to define zipcode sequences have taken advantage of their independent and modular nature, i.e., fusion of an RNA FEBS Letters 592 (2018) ª 2018 Federation of European Biochemical Societies 2951

5 RNA localization: functions and disease links S. Bovaird et al. A B C D Fig. 1. mrna localization is observed across many species and cell types. Examples of: (A) ash1 localization to the bud tip in yeast (B) osk, grk, and bcd localization in the Drosophila oocyte (C) vg1/vgt localization in the Xenopus oocyte (D) b-actin localization to the cell extremities of fibroblasts in the chicken. zipcode can typically impart specific localization behavior to a normally unlocalized reporter RNA. Such studies have revealed that zipcodes can be defined by their sequence and/or structural features, either spanning from just a few up to several hundred nucleotides [83 85]. For instance, during Drosophila development many transcripts with particular zipcode elements in their 3 0 UTR, including nanos, bicoid, and oskar, serve to pattern the anterior-posterior axis [28,82,86 88]. In the case of bicoid (bcd), a 437- nucleotide fragment in its 3 0 UTR containing five stem loops is involved in localizing this transcript to the anterior pole of the oocyte/embryo [86]. This localization is achieved through the binding of the trans-acting protein complex ESCRTII to the 3 0 UTR to mediate microtubule-dependent transport, followed by anchoring in the anterior pole through interaction with the RBP Staufen and bcd stem loops III, IV and V [89 91]. In another example, wingless (wg) mrna is selectively targeted to the apical cytoplasm of Drosophila embryos via a 53-nucleotide WLE3 stem loop in its 3 0 UTR [92]. This sequence shares similarity with hairy, ftz, K10, and orb stem loop elements, and is evolutionarily conserved across 13 species of Drosophila [92 94]. By contrast to the transcripts mentioned above, mrnas can foster zipcode elements within their 5 0 UTR or coding region, some containing multiple elements dispersed throughout their sequence, as described for Ash1 mrna in yeast [83,95]. A similar logic holds for the localization of noncoding RNAs. For example, MALAT1, a nuclear lncrna, utilizes signals at its 5 0 and 3 0 extremities to direct its localization to paraspeckles, an RNA granule thought to be implicated in transcriptional regulation and nuclear retention of edited RNA [96]. These cases highlight how spatial RNA targeting can be influenced by zipcode elements, which can be distributed across the native RNA transcript and act alone or in tandem (Fig. 2A). Classic zipcode mapping studies utilizing transgenic reporter RNA systems have proven powerful, yet are highly time consuming. However, such efforts should be aided with the emergence of massively parallel assays exploiting next-generation sequencing methods to characterize RNA regulatory elements, including those involved in localization control [97 99]. Such is the case in a notable study by Shukla et al. who tested over sequences derived from spatially-distributed lncrnas, which were individually inserted into the 3 0 UTR of a frame-shifted Sox2 mrna, a strategy that 2952 FEBS Letters 592 (2018) ª 2018 Federation of European Biochemical Societies

6 S. Bovaird et al. RNA localization: functions and disease links enable them to identify a panel of RNA segments that could enhance the nuclear enrichment of a normally cytoplasmic reporter transcript [98]. Using a similar strategy, Lubelsky and Ulitsky were able to uncover a 42-nt sequence derived from transposable element RNAs, designated SIRLOIN (SINE-derived nuclear RNA localization), which drives RNA nuclear localization through association with the HNRNPK protein [99]. In addition to such high-throughput experimental assays to help define zipcode elements, computational methods will be crucial in characterizing the primary sequence and structural features of putative zipcodes. A number of computational methods have been developed to identify potential cis-acting motifs from RNA datasets, such as MEME, HOMER and SeAMotE [ ]. Moreover, numerous prediction software have emerged that computationally evaluate RNA secondary structure features [ ]. These analyses hold the potential to be further compared to experimental approaches to uncover zipcode elements otherwise glossed over by sequencefocused methods alone. Trans-acting RNA localization regulatory elements RBPs make up a broad class of proteins that interact with RNAs at every step of their metabolism, including 5 0 and 3 0 end processing, splicing, nuclear export, intracellular trafficking, translation, and stability regulation. As a regulatory class, these factors are among the most deeply conserved evolutionarily [113,114], and they can be subclassified according to the types and combinations of RNA-binding domains (RBDs) they contain [115]. These RBDs serve to mediate binding to primary sequence or structural features of target RNAs (Fig. 2B). Many RBDs, such as KH, DEADbox, RRM, and S1 domains, bind single-stranded RNA (ssrna) in a sequence-specific manner [ ]. For instance, the trans-acting factor IGF2BP1/ ZBP1 contains four KH domains which together increase binding capability of zipcode elements in the 3 0 UTR of b-actin mrna [74]. Additionally, ZBP1 has two RRM domains that permit localization of this transcript along axonal projections [74]. Clearly, the occurrence of multiple RBDs can serve to confer greater specificity to the consensus sequences an RBP interacts with. Other binding domains preferentially recognize RNA structure, such as double-stranded RNA (dsrna) regions, which are appropriately dubbed dsrbds [122]. Unlike RRM domains, the majority of intermolecular contacts imparted by dsrbds are in the phosphate backbone of RNA helices and, as such, they recognize RNA shape rather than RNA sequence [122,123]. For example, the RNAediting enzyme ADAR1 contains three dsrbds, one of which acts as a dsrna-binding-dependent nuclear localization signal [124]. Here, the RBD serves as both a contact interface to allow assembly of a ribo-nucleoprotein (RNP) complex and as a targeting signal itself. Thus, RBDs interface with RNA transcripts via their cis-acting elements to form RNPs and may act independently or combinatorially with other modular domains. While RBPs have long been known to play critical cellular housekeeping functions, several innovative approaches implemented over the last decade have dramatically expanded the repertoire of known RBPs, while also providing systematic platforms to characterize their binding properties, both in vitro and in vivo. Indeed, RBP interactome capture studies, involving the mass spectrometry (MS)-based characterization of proteins crosslinked to polyadenylated (polya) or nonpolya RNA, have established a staggering repertoire of > 1500 RBPs, including many enigmatic proteins with no obvious RBDs based on sequence and structural homology searches [ ]. Such approaches have also been complemented by algorithmic methods that can predict RPBs from protein protein interaction datasets, since proteins that associate with known RBPs are more likely to be RBPs themselves [129]. To define the consensus sequence (and potentially structural)-binding motifs or RBPs, several systematic in vitro selection approaches have been developed in which purified RBPs are first incubated with randomized RNA mixtures, then RBP-bound transcripts are purified and characterized via microarray or RNAsequencing (RNA-seq) approaches [121, ]. Additional methods have been developed to map protein RNA interactions in vivo in a cellular model of interest, in particular UV crosslinking and immunoprecipitation of selected RBPs combined with highthroughput sequencing of their associated transcripts [ ].Computational tools may be partnered with such in vitro and in vivo-binding datasets, along with further in vitro assays, to unearth distinct regulatory features bound by given RBPs [130, ]. In addition to these RBP-oriented methods, alternative RNA-centric approaches have been developed to identify specific proteins that interact with a given RNA transcript. This includes the use of RNA-capture methods, in which one can selectively purify a target RNA using antisense capture probes, followed by MS profiling of associated proteins [ ]. Interestingly, Ramanathan et al. [150] have recently developed a method using BirA-mediated proximity labeling paired FEBS Letters 592 (2018) ª 2018 Federation of European Biochemical Societies 2953

7 RNA localization: functions and disease links S. Bovaird et al. with a kn-boxb targeting system to selectively biotinylate and purify proteins that associate with an RNA of interest in live cells. While these approaches are used more broadly to interrogate RNA protein interactions, without a specific focus on RNA localization, they are crucial in helping to identify RPBs that are likely to be implicated in RNA maturation and trafficking. As mentioned above, RBPs are not the only regulatory molecules that can influence post-transcriptional gene expression events in subcellular space; in particular, noncoding RNAs such as lncrnas or mirnas may also participate in this process (Fig. 2B). These factors, which can harbor their own zipcode sequences controlling subcellular targeting, may also work in trans to regulate the functional properties of mrnas within specific subcellular locales or to help mediate their subcellular targeting by providing a scaffolding function. Indeed, lncrnas themselves experience specific subcellular localization in accordance with their cis-acting sequences [61,63,98,99,151,152]. However, they also function in trans by acting as scaffolds for chromatin remodeling enzymes. Such is the case of Xist, an essential regulator of X-chromosome inactivation. Using the comprehensive identification of RNAbinding proteins by mass spectrometry (ChIRP-MS) method, 81 protein interactors of Xist were identified including HNRNPU, involved in its localization, and HNRNPK, which mediates gene silencing [146]. LncRNAs may also serve as architectural components by proving recruitment sites for RNA transcripts in a range of cytoplasmic and nuclear RNA granules (Fig. 2C), as has been shown for Neat-1 in paraspeckle formation [ ]. On the other hand, mirnas constitute part of the mirisc complex, along with their associated effectors, Argonaute proteins, another trans-acting-binding partner [ ]. Loaded mir- NAs bind target transcripts through partial or full antisense base pairing interactions to destabilize or silence their translation [ ]. Many mirnas are specifically targeted to subcellular structures within the cell, as reviewed by Leung and Sharp [164]. For instance, they can associate to the endoplasmic-reticulum to recruit RNAs translated by ER-bound polysomes [165]. Moreover, some mirnas can harbor short zipcode elements themselves, as was demonstrated for mir29b, which contains a zipcode modulating its nuclear targeting [166]. Using single molecule localization microscopy, mirnas have also been shown to localize to sites of exosome biogenesis [167]. Recent studies have demonstrated that mirnas experience differential subcellular distribution in neurons [ ]. For example, mir-124 regulates axonal pathfinding and neural maturation by silencing transcription factor expression, such as that of Sox9, exclusively in the cell soma [170,172]. Furthermore, mir-124 has been shown to act as a linear timer during neural development by controlling sensitivity to semaphorin Sema3A [173]. Conversely, mir-26a is present in dendrites to target MAP2 mrna, whose protein product is selectively localized in dendrites [170,174]. Indeed, many mirnas have also been shown to modulate mrna translation in dendritic and axonal projections in response to external stimuli [20,64, ]. As highlighted here, both noncoding RNAs and RBPs may interact with mrna transcripts in trans to seed mrnp formation, priming recruitment of effectors to carry out complex regulatory functions including but not limited to subcellular targeting. Beyond the mrnp: RNA granules and their biological functions Membrane-less cellular bodies, including structures such as nucleoli, germ granules, processing bodies (Pbodies), stress granules, and paraspeckles, have emerged as hotbeds of post-transcriptional regulation of gene expression in different organisms and cellular states [154,164, ]. Like RNA transport granules discussed above, these structures are thought to provide isolated chemical environments, teeming with select RNA species and RBPs (e.g., helicases, decays enzymes, translation factors), in which specific regulatory processes can take place with higher efficiency (Fig. 2C) [153,179, ]. For example, paraspeckles are nuclear granules that have been implicated in the nuclear retention of A-to-I edited mrna transcripts [181], whereas cytoplasmic P-bodies are sites of mrna degradation or storage depending on the context [187,188]. While these granules are present under steady state conditions, other membrane-less structures are formed in response to stress. Indeed, SGs are structures formed in the cytoplasm in response to various stresses (e.g., oxidative stress, heat shock) that sequester and stabilize mrnas, RBPs and translation initiation factors in a manner that depends on the stress context [ ]. Importantly, as will be discussed below, these granules exhibit altered biogenesis dynamics in specific diseases, thus underlining their importance as hubs for the coordination of cellular activities. For RNA granules to maintain their function throughout various cellular contexts, they must ensure that both cis- and trans-factors are targeted and retained in their membrane-less bodies while promoting dynamism with the intracellular milieu. This targeting method is reliant on a resurgent physicochemical 2954 FEBS Letters 592 (2018) ª 2018 Federation of European Biochemical Societies

8 S. Bovaird et al. RNA localization: functions and disease links mechanism based on liquid-liquid phase separation, a process involving the de-mixing of two immiscible liquids from one another due to each holding differential components or properties, much like oil in water [193]. Liquid phase transitions share many common physiognomies, illustrated through studies of liquid droplets both in vivo and ex vivo [178, ]. One excellent model of these shared features is the P granule, the first cellular body characterized as a liquid droplet by Brangwynne and collaborators. These RNA granules form within Caenorhabditis elegans single-cell embryos, where they localize to the posterior pole and thereby serve to promote differentiation of progenitor germ cells [178,198]. Moreover, the authors showed that P granules are spherical, appear to drip off solid surfaces and can also fuse together upon contact [178]. Known RBP components of various RNA granules, such as FUS, LAF-1, TDP-43, and HNRNPA1 may also form droplets that exhibit wetting independently in vitro [196,199,200] and can exchange components dynamically with their surrounding milieu [178, ]. Initiation of RNA granules by phase transition occurs through a combination of means, all dependent on molecular interfaces reliant on both RNAs and proteins (Fig. 2C). Interaction of proteins through intrinsically disordered regions (IDRs) and repetitive folded motifs promotes formation of a condensed liquid phase through electrostatic and hydrophobic interactions between aromatic and positively charged residues common to low complexity domains [180,193,200,205,206]. For example, DEAD-box helicases Ddx4, LAF1 and HNRNPA1 contain IDRs within their RNA recognition motifs that trigger phase separation in germ granules, P granules and stress granules, respectively [195,196,200,202]. RNA granules may also be seeded through the oligomerization of RNA transcripts within mrnps via their cis-acting sequences, causing large scale recruitment of other factors to the emerging phase, as is documented in the case of oskar mrna transport granules in fly oocytes/ embryos [69]. lncrnas can also play a very specific role in constructing granules [153]. For example, lncrna NEAT1 tightly localizes to nuclear paraspeckles and acts as an architectural scaffold within the granule, mediating molecular interactions [154,207]. This is dependent on FUS, in particular its prion-like domain, a phase-transition promoting IDR [205, ]. This phenomenon was explored by Chujo and colleagues by exploiting the semi-extractible nature of lncrnas and designing a protocol to undercover their hidden localization and architectural roles in a plethora of membrane-less granules [153,208]. The construction of RNA granules through condensation of a liquid phase, and their continued aggregation, is clearly reliant on RNP components and the proteinprotein and RNA protein interactions inherent to them. Liquid-liquid phase transition exhibited in RNA granules offers a new look at complex targeting mechanisms employed by the cell to localize transcripts based on fate and function in both normal and stress contexts. Connections to disease In addition to the important functions fulfilled by RNA localization pathways under steady-state or stress-induced conditions, mounting evidence indicates that perturbations of these regulatory events can lead to disease emergence. Indeed, aberrantly localized coding and noncoding RNA species are increasingly being recognized as important molecular lesions associated with disease and there is a growing need to understand how these deviant pathways arise [211]. Below, we discuss examples of how disturbances in the different components involved in RNA trafficking represent critical events in the emergence of neuromuscular disorders and cancer. Moreover, a growing body of literature is underlining the disease relevance of the process of extracellular vesicle (EV)-mediated transfer of RNAs beyond the confines of individual cells. Neuropathologies The etiology of several neurological diseases has been primarily characterized by the abnormal accumulation of protein aggregates that manifest as extracellular senile plaques or as intracellular inclusions confined to the cell bodies, nuclei and processes of neurons [212]. As the aforementioned would suggest, neuropathogenesis has been historically attributed almost exclusively to deviant protein behavior, but increasing evidence suggests that a defining feature of these pathologies lies at the hands of anomalous RNA localization routes [213,214]. For example, a common theme in neurological and neuromuscular disorders is the presence of unstable microsatellites in the form of short nucleotide repeat expansions (SNRs) in genes, that once transcribed, lead to the mislocalization and accumulation of these repeat-containing gene products [215]. The size of these SNRs can be of significant variability, but their pathogenicity is more pronounced with larger repeat sizes [216]. The processes behind the neurodegenerative effects of these SNRs are attributed to two main proposed scenarios: the loss-of-function model, where the repeat expansions perturb the normal function of the gene they reside in [217,218]; and FEBS Letters 592 (2018) ª 2018 Federation of European Biochemical Societies 2955

9 RNA localization: functions and disease links S. Bovaird et al. a toxic gain-of-function model, where the SNRs lead to the aberrant functional gains of the encoded gene products. The latter can be described by two main mechanisms: first, the SNR-containing RNA may exert a toxic gain-of-function phenotype by forming abnormally-localized nuclear aggregates that bind and sequester select RBPs, thus disrupting normal localization pathways (Fig. 3. Steps 1 4) [219,220]; second, SNR transcripts can be translated into toxic dipeptide repeat (DPR) proteins (Fig. 3. Steps 5 6) [221,222]. Myotonic Dystrophy (DM), the most common form of muscular dystrophy, is a dominantly inherited multisystemic disease that entails a diverse set of symptoms, including myotonia, intellectual disability, cataracts and cardiac defects [223]. This form of muscular dystrophy manifests itself through two forms: DM1, which results from an expanded CTG repeat in the 3 0 UTR of the DMPK gene; and DM2, characterized by a CCTG expansion in intron 1 of the ZNF9 gene [ ]. The molecular mechanisms conferring the pathogenicity of these repeat expansions are likely multifaceted, involving abnormal RNA localization and translation defects. For instance, in both DM1 and DM2, aberrantly expressed CUG and CCUG repeat RNAs aggregate within subnuclear bodies that act as sponges by binding and sequestering multiple RBPs, such as members of the Muscleblind (MBNL) family of proteins (Fig. 3. Steps 1 2) [227,228]. MBNL proteins belong to a family of splicing factors that regulate mrna localization and alternative splicing, including essential splicing events implicated in the development of skeletal and cardiac musculature [229,230]. Hence, the sequestration of these proteins by toxic repeat-containing RNA plays a crucial role in the pathology of DM, especially regarding misregulated splicing events [ ]. Additionally, a recent cell fractionation-based transcriptome-wide analysis of RNA subcellular localization of cells depleted of MBNL proteins revealed broad alterations in mrna subcellular distribution patterns [49]. Finally, deviant protein translation has been reported in DM via a process involving repeat-associated non-atg-dependent (RAN) translation, resulting in variable homopolymeric proteins of a potentially toxic nature (Fig. 3. Steps 5 6) [233,234]. Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disorder, which is characterized by rapid and progressive paralysis leading to respiratory failure and death [214]. This disease is of multifactorial nature and several molecular pathways have been A C B Fig. 2. Interaction of cis-acting sequences and trans-acting regulatory partners in localized RNA transcripts. (A) Cis-acting sequences, or zipcodes, in red, reside within the transcript and serve to interface with other factors to subcellularly target RNAs. Zipcodes may interact with their partners via sequence and/or shape (ex. stem loop secondary structure). (B) Trans-acting-binding partners interface with RNAs to help confer subcellular targeting. RBPs bind cis-sequences through modular RNA-binding domains, and may act in tandem, with potential to have many bound per transcript. mirnas, as part of the mirisc complex, contact complimentary sequences in the target. lncrnas may interact with complimentary sequence and/or secondary structure of the RNA. (C) Membrane-free RNA granules are formed via liquid liquid phase transition, and contain specifically targeted RNAs, through cis- and trans-factors, along with other regulatory molecules to dictate transcript fate. lncrnas often serve as an architectural scaffold for granules, contacting many components in tandem FEBS Letters 592 (2018) ª 2018 Federation of European Biochemical Societies

10 S. Bovaird et al. RNA localization: functions and disease links attributed to its onset [ ]. The most common cause of ALS is ascribed to a GGGGCC (G 4 C 2 ) hexanucleotide repeat expansion within the C9orf72 gene [221,235]. C9orf72-G 4 C 2 repeat sequence length varies among ALS patients, although the disease emergence is usually observed in patients with > 30 repeats [ ]. The molecular mechanisms of pathogenesis of C9-ALS appear to be conferred by the toxic gene products resulting from repeat-associated transcription and translation. Similar to the situation in DM1/2, the G 4 C 2 repeat RNA products (and the antisense transcribed C 4 G 2 ) form nuclear aggregates that appear to sequester RBPs and hence may lead to profound perturbations in posttranscriptional regulation (Fig. 3. Steps 1 2) [239,240]. Moreover, these repeat transcripts can escape the nucleus and undergo RAN translation, ultimately leading to the aggregation of aberrant polydipeptide proteins that can dramatically alter the nucleocytoplasmic shuttling of cellular molecules (Fig. 3. Steps 5 6) [240,241]. Many neurological disorders, including ALS, are characterized by the presence of insoluble proteinaceous aggregates in neuronal cells of affected individuals, which include RBPs and RNAs as key components (Fig. 3. Steps 1 6) [242,243]. The mechanisms controlling the formation of these aggregates remain incompletely understood, but mounting evidence suggests that this is linked to the ability of RBPs and RNAs to form phase-separated nonmembranous structures, such as SGs [200,244]. For instance, inherited forms of these disorders can be caused by missense mutations in RBPs, such as TDP-43, FUS/TLS and hnrnp A2/B1 [ ], producing SGs with altered composition, subcellular localization and dissociation dynamics [245, ]. Interestingly, repeat expansion transcripts have been shown to provide a template for the multivalent base-pairing required for the phase transition of these RNAs into a gel-like state without the need of protein components [251]. More recently, compelling new studies have highlighted the importance of RNA secondary structure as an essential driving force in the establishment of phase separation behavior, where RNAs importantly regulate the biophysical properties of RBPs [210,252]. These intriguing phenomena emphasize recurring themes which are likely operating in similar ways in many neuromuscular disorders [ ], and in so doing underscore the importance of abnormal localization and aggregation behavior of RNAs and the proteins that bind to them. Hence, treatments for these diseases should incorporate the targeted disruption of these mislocalized RNAs through innovative therapies such as the use of synthetic antisense oligonucleotides (ASOs), which can alter the activity and stability of transcripts through diverse mechanisms [256,257]. Links to cancer Cancer encompasses a heterogenous group of diseases that in unison portray features of malignant transformation, such as abnormal and uncontrolled cell growth, replicative immortality, activation of invasion and metastasis, induction of angiogenesis, and resistance to cell death [258]. These types of neoplastic diseases are complex and multifactorial, depending on specific regulatory networks that dictate their progression, survival and malignancy. Although traditionally viewed as disorders of aberrant regulation in transcription and signaling events, alterations in RNA regulation behavior are coming to light as potential players in neoplasia [259,260]. For instance, recent evidence has emerged that proteins involved in RNA localization are aberrantly expressed in tumors, suggesting that irregular RNP-pathways might be important players in the larger underbelly defining cancer pathophysiology [ ]. In a recent study analyzing mutations in 1344 RBP encoding genes across > 6000 cancer samples corresponding to 26 cancer types, a subgroup of 281 RBPs were found to be enriched for mutations in at least one sample [260]. Functional analysis of these RBPs found that they were enriched in pathways associated with apoptosis, translation and splicing, highlighting possible roles in cancer progression and mirroring previous findings [260,262]. A well-studied example of an RBP with oncogenic activity is SRSF1 [ ], which has been linked to the splicingmediated regulation of many cancer-associated genes [269,270], but is thought to play diverse roles in RNA metabolism ranging from transport to translation [271,272]. Post-transcriptional regulation of RNA plays an important role in tumorigenesis [273,274]. The means by which these regulatory events may lead to the development of cancer has not been fully defined, nevertheless, several RBPs have been linked to malignancy [274,275]. A noteworthy example of this occurrence is one that involves the insulin-like growth factor 2 mrna-binding (IGF2BP) family of oncofetal proteins, an ancient and evolutionarily conserved family of RBPs that participates in various facets of cellular function, including cell polarity, morphology, migration and proliferation [ ]. IGF2BP-members are expressed during embryonic development, with relatively lower levels detected in adult tissues [229,276,281]. Remarkably, the increased expression of these RBPs has been identified in varied malignancies FEBS Letters 592 (2018) ª 2018 Federation of European Biochemical Societies 2957

11 RNA localization: functions and disease links S. Bovaird et al. [ ], and have been correlated with poor prognosis in ovarian cancer [284]. The mechanisms through which IGF2BPs might contribute to cancerogenic transformation is probably multilayered, as more than a 1000 different mrnas have been found to be bound by these factors in genome-wide studies [285,286]. A classic example of these transcripts is b-actin mrna, for which the transport and localized translation at the leading edge of cells is regulated by IGF2BP1/ZBP1 [287]. These findings provided insights into earlier studies, where the normal subcellular localization of this mrna was found to be altered in metastatic cancer cell models to a more perinuclear distribution [288], suggesting mislocalization in this mrna in this context. In another example, IGF2BP proteins involvement in cancer, it was found that in transformed cells, IGF2BPs reside in RNP granules targeted to invadopodia, cell-protrusions thought to degrade the extracellular matrix, which could potentially drive metastatic dispersion [289]. These assorted mechanisms suggest that RNA localization pathways may affect various aspects of cancer progression. Interestingly, invadopodia have recently been proposed as important docking sites of exosome-containing multivesicular bodies, showing a synergistic relationship between extracellular vesicles (EVs) and these cellular projections during cancer progression [290,291]. Extracellular vesicles: emerging concepts Extracellular vesicles (EVs) represent a highly heterogenous group of membrane-bound nanoparticles that are released by cells to the extracellular space. These nanoparticles contain a diverse array of bioactive molecules including proteins, signaling molecules and RNA [ ], and have recently come to the forefront as novel mediators of intercellular communication in both healthy and disease contexts [ ]. Upon release from their cells of origin (Fig. 4), EVs act as molecular shuttles whereby they have the capacity to travel through the extracellular environment, then fuse with and deliver their contents to recipient cells. After their release into the cytoplasmic space, these contents are then able to induce phenotypic changes which alter the normal behavior of recipient cells [292]. The EV-content repertoire is representative of their cells of origin, yet they contain particular types of molecules that are over represented Key DNA Transcrip on factors RNA Polymerase RNA Molecule RNA Binding Protein RNA RBP Granule Ribosome Nuclear pore Polydipep de proteins Protein aggregates Fig. 3. RNA localization and neurological disorders: (1) RNA is transcribed from genomic DNA via RNA Polymerase II in association with transcription factors (TFs). (2) Aberrantly-spliced or unspliced RNA transcripts containing short nucleotide repeat expansions (SNRs) act as sponges, sequestering multiple RNA-binding proteins (RBPs) forming nuclear foci in a toxic-rna model. (3, 4) SNR-containing transcripts are exported from the nucleus to the cytoplasmic space, where they are mislocalized in the cytoplasmic space and then associate with RBPs forming RNP-containing granular lesions. (5, 6) Alternatively, SNR-containing RNA may undergo a form of deviant translation known as repeat-associated non-atg-dependent (RAN) translation, resulting in potentially toxic polydipeptide proteins. These proteins may then abnormally aggregate, forming characteristic insoluble proteinaceous lesions FEBS Letters 592 (2018) ª 2018 Federation of European Biochemical Societies

12 S. Bovaird et al. RNA localization: functions and disease links Key Early endosome 1 DNA Transcrip on factors Mul vesicular endosome 3 Exosomes 6 9 RNA polymerase Transcribed RNA RNA binding protein Nuclear pore Mature RNA Exosome Microvesicles Ribosome 7 Microvesicles 8 Translated proteins Posttransla onal modifica on Fig. 4. RNA localization and sorting to sites of extracellular vesicle biogenesis: (1 3) RNA is transcribed from genomic DNA via RNA Polymerase II in association with transcription factors (TFs). Next, RNA-binding proteins interact with nascent RNA transcripts in various maturation processes including splicing, 5 0 -capping, polyadenylation and folding. Following various maturation steps, mature RNA is exported from the nucleus via nuclear pores to the cytoplasmic space, where its life cycle may proceed. (4 6) Subsets of RNA transcripts are selectively recruited to sites of exosome-biogenesis via the use of post-translationally modified RBPs (e.g., SUMOylated hnrnpa2b1 [303]). These RNAs are incorporated into endosomal bodies via invagination processes that lead to the formation of intraluminal vesicles (ILVs) sequestered within multivesicular endosomes (MVEs). Next, MVEs fuse with the cell membrane and release exosomes to the extracellular space. (7, 8) Subsets of RNA transcripts are selectively recruited to sites of microvesicle-biogenesis via the use of post-translationally modified RBPs. Microvesicles or shedding bodies arise from the outward blebbing of the cell membrane. Next, a fission process leads to the release of these microvesicles to the extracellular milieu. (9) Alternatively, RNA may not be sorted to these vesicular bodies at all and is rather transported to various cellular subcompartments for localized translation of their encoded proteins. from those of their parental cells, including both coding and noncoding species of RNA [294]. In fact, growing evidence suggests that the RNA profiles of EVs can be dramatically different between cell types, and even between subpopulations from the same cell [301]. The mechanisms behind the recruitment of these RNA molecules to EVs is likely to be intricate and to involve the interplay of RNA cis-acting elements and trans-acting RBPs. These RNA RBP interactions form RNPs that dictate the localization routes of RNA populations to sites of EV-biogenesis [302]. Although these mechanisms remain relative obscure, evidence has come forth suggesting that certain post-translational modifications of the RBPs involved may be required (Fig. 4. Step 3) [292,303]. The diversity of RNA species identified in EVs comprise commonly studied mrnas, mirnas, and rrnas, as well as more elusive short noncoding RNA, lncrna, circular RNA, trna fragments, vault RNA and Y-RNA [294, ]. This characteristic variety of RNA molecules highlights the potentially wide-ranging signaling pathways they may affect. The incorporation of these RNA molecules into EVs may very well present itself as a protective and efficient means of transferring these genetic signals to local and distant cellular microenvironments, and as such may be an essential component in the cross-talk required for the spread of certain diseases. Although not yet fully understood, the impact of RNA localization to EVs is becoming apparent. For example, in cancer models, EV-mediated transfer of different RNA molecules has been found to induce diverse phenotypes including the stimulation of cell proliferation, angiogenesis, and metastasis [298,307,308]. For instance, mrnas encoding for the notorious and highly tumorigenic epidermal growth factor receptor (EGFR) FEBS Letters 592 (2018) ª 2018 Federation of European Biochemical Societies 2959