Intracellular Trafficking of RNA in Neurons

Transcription

1 Traffic 2006; 7: Blackwell Munksgaard Review # 2006 The Authors Journal compilation # 2006 Blackwell Publishing Ltd doi: /j x Intracellular Trafficking of RNA in Neurons Wayne S. Sossin 1, * and Luc DesGroseillers 2 1 Department of Neurology and Neurosurgery, McGill University, Montreal Neurological Institute, BT 110, 3801 University Street, Montreal, Quebec, Canada H3A 2B4 2 Départment de Biochimie, Université de Montréal, Montréal, Quebec, Canada H3T 1J4 *Corresponding author: Wayne S. Sossin, wayne.sossin@mcgill.ca The transport of messenger RNAs (mrnas) in neurons serves many purposes. During development, trafficking of mrnas to both axonal and dendritic growth cones regulates neuronal growth. After synapse formation, mrnas continue to be transported to dendrites both as a mechanism for the localization of proteins to specific compartments and as a substrate for local translational regulation of synaptic plasticity. Finally, activity-dependent mrnas are transported quickly to dendrites after transcription. Determining how mrnas are transported and specifically translated in these different paradigms is a major unanswered question. Addressing this question is also complicated by the presence of many other RNA processing and storage centers that may not be involved in transport but share components with the transport structures. In the present review, we will discuss several recent studies addressing mechanisms of mrna transport in neurons, as well as proteomic characterization of mrna transporting structures in neurons. We define two types of RNA transport structures in neurons, transport particles and RNA granules and distinguish them by the presence or absence of ribosomes. We will present a number of different molecular models for how mrnas are repressed during transport, and how these may affect the regulation of local translation in neurons. Key words: micro RNAs, P bodies, RNA granule, Staufen, stress granules, translation, transport particle Received 17 July 2006, revised and accepted for publication 22 September 2006, published on-line 20 October 2006 RNA transport is of particular importance to neurons due to the complexity of their intracellular compartments and the use of local translation in these compartments for local regulation of growth and synaptic strength. Examination of RNA transport in neurons largely consists of examining dendritic spots (or puncta) containing either a specific RNAbinding protein or a messenger RNA (mrna) (often an artificial mrna containing MS2 sites visualized by a green fluorescent protein MS2 fusion protein). It is important to realize that these puncta could be any of several types of cytoplasmic structures (see below). Most co-localization studies are confounded by the presence of many RNAbinding proteins in multiple types of cytoplasmic RNA structures (Table 1), probably because mrnas transit through these structures in a dynamic fashion (Figure 1). There are Many Different Cytoplasmic RNA Structures in Neurons For clarity, we first define the different cytoplasmic macromolecular RNA structures that exist in neurons: translating polysomes, processing bodies (P bodies), stress granules, micro RNA particles (mirnps) or the RNA interfering silencing complex (RISC), transport particles and RNA granules. Many of the RNA-binding proteins and other proteins discussed in this manuscript may play roles in multiple macromolecular RNA structures. Moreover, mrnas transport dynamically between these compartments and understanding these dynamics will be important in understanding RNA localization (Figure 1). We will use studies from diverse organisms from yeast to Drosophila to mammals, assuming that certain aspects of RNA trafficking and cytoplasmic RNA structures are highly conserved (1). Translating polysomes The classic macromolecular RNA-containing structures are translating polysomes. These contain many RNA-binding proteins, ribosomes and mrnas and may be difficult to distinguish from other RNA structures such as RNA granules. Indeed, two distinct studies suggest that mrnas in RNA granules can convert into translating polysomes after neuronal stimulation (2,3). P bodies Processing bodies are sites for storage of repressed mrnas and for mrna degradation. The P bodies contain proteins involved in mrna degradation such as decapping enzymes and endonucleases (4). The P bodies are best characterized in yeast where two proteins, Ddh1 and Pat1b, have been particularly implicated in their formation because overexpression of these proteins lead to increases in the number of P bodies while loss of these proteins leads to loss of P bodies (5). A model proposed by Parker and colleagues is that proteins like Ddh1 act as both general repressors of translation and as adaptors that bring mrna to P bodies to induce decapping. Indeed, Ddh1 physically interacts with decapping enzymes and stimulates 1581

2 Sossin and DesGroseillers Table 1: Listing of the localization of proteins discussed in the text to different types of RNA containing structures. Some data comes from a similar table recently published by Anderson and Kedersha (33) and other studies are referenced in the text. P bodies Stress granules RISC complex Polysomes RNA granules Transport particles Staufen 1 Yes Yes ND Yes Yes Yes Staufen 2 ND Yes ND Yes Yes Yes Ddh1/Rckp54/DEAD box 6 Yes Yes ND ND Yes ND Decapping enzymes Yes No ND No No ND G3BP No Yes ND Yes Yes ND EIF4E Yes Yes ND Yes No Yes CPEB Yes Yes ND ND ND Yes ZBP ND ND ND ND Yes Yes FMRP Yes Yes Yes Yes Yes Yes TIA-1/R Yes Yes No No No No Pumilio No Yes ND ND ND Yes Poly(A)-binding protein No Yes Yes Yes Yes Yes Small ribosomal proteins No Yes May be Yes Yes No Large ribosomal proteins No No May be Yes Yes No Argonaute ND Yes Yes ND ND ND ND, not determined. their activity (6). In P bodies, mrnas are not associated with ribosomes; indeed, ribosomes and P bodies compete for mrnas and repression of translation initiation may be sufficient to target mrnas to P bodies (7). As mrna turnover is likely to be important at synaptic sites, it is likely that P bodies will be found in dendrites (8). Moreover, as transported mrnas are also repressed, there may be a relationship between transport particles in neurons and P bodies in other cells. Stress granules Stress granules are induced by the phosphorylation of eif2a (9,10), which prevents mrnas already bound to the small 40S ribosomal subunit from initiating translation. These mrnas, associated with the small ribosomal subunit, are then stored in a stress granule awaiting the disappearance of the stressor and renewal of translation (11). A number of specific mrna-binding proteins such as G3BP, Pumilio and TIAR (or TIA-1) have been suggested to be key components of this structure (8,12,13). The mrnas in stress granules may be removed from the small subunit and then sent to P bodies that are proposed to physically associate with stress granules (14). The eif2a phosphorylation levels may be high enough in neurons to have stress granules at rest (8). Stimulating neurons to induce long-term potentiation leads to dephosphorylation of eif2a (15), and thus repressed mrnas may be released through this mechanism. RISC complex or mirnp complex The mrnas actively repressed by micro RNAs (mirnas) are found in a mirnp, also known as a RISC complex. Components of the RISC complex include argonaute proteins, TRBP, DICER, PACT and the fragile X mental retardation protein (FMRP) (16,17). The RISC complex can be co-localized with P bodies and this may lead to degradation of mrnas through mirnas. Whether or not mirnas bind to ribonucleic proteins (RNPs) or ribosomebound mrnas is controversial (18). Some evidence suggests that mirnas only block translational initiation of capped mrnas, suggesting a block in ribosome recruitment (19,20). However, mirnas are found associated with ribosomes, especially in the neurons, and recent evidence suggests that they can also block translation at the elongation step (21,22). However, mirnas, while present on polysomes in brain, were excluded from RNA granules (21). Figure 1: (Opposite) Dynamics of RNP travels in a neuron. The green circle represents the cap on all mrnas. Square boxes represent RNA-binding proteins that remain associated with an mrna through its life cycle. Red octagons represent translational repressors (either through initiation or elongation). Blue crosses represent adaptor proteins. Black ovals are 80S ribosomes. Two-way arrows indicate that an mrna can travel both ways (i.e. from an RNA transport particle to a P body and back again). One-way arrows indicate one-way transport (i.e. mrnps can go from a stress granule to a P body but cannot re-enter a stress granule without regaining initiation factors). After exit from the nucleus, a ribonucleic particle has two major choices. Either it will become initiated by binding of eif4e and eif4g or bind to a translational repressor. After binding to a repressor, it can either be targeted to a P body for degradation or if containing the appropriate signals for binding to unknown adaptors, be targeted to an RNA transport particle which using a microtubule motor can be sent to axons or dendrites. The mrnas that are repressed by mirnas will be associated with the RISC complex, which may help target the mrna to a P body or to an RNA transport particle. This association may occur either before or after transport. After the first step of translation initiation, the RNP can continue on to translation, or if eif2a phosphorylation levels are high, be stored in a stress granule. After polysomes have begun translation, through an unknown mechanism, neurons can form an RNA granule from the polysomes that will allow for the blockade of elongation and the transport of the mrna to axons or dendrites. Upon de-repression, the RNA granule will turn back to a translating polysome Traffic 2006; 7:

3 RNA Trafficking in Neurons Transport particles We are defining two types of RNA transport based on whether or not transport is associated with ribosomes. Transport particles are macromolecular structures that transport mrnas in the absence of ribosomes. These complexes contain (i) mrnas; (ii) specific RNA-binding proteins; (iii) adaptors that connect the RNA-binding protein to a motor complex; (iv) motors and (v) additional RNAbinding proteins that are important for the repression of translation before reaching the correct target and anchoring the mrna at the target. The best characterized transport particle is the complex transporting the ASH1 mrna in yeast consisting of the mrna-binding protein (She2P), an adaptor that binds to both the RNA-binding proteins and motors (She3P), translational repressors, including a Pumilio-like proteins, anchoring proteins (She5P, Bud6p) and a myosin motor (She1P) (23 25). This particle has been purified and about 22 distinct mrnas are carried by Traffic 2006; 7:

4 Sossin and DesGroseillers this particle (26). Importantly, the first step in formation of the transport particle may be the translational repression of the ASH1 mrna (23). While transport particles are important for transport in neurons as well and many specific RNA-binding proteins and translational repressors have been identified (see below), there have only been partial characterizations of any particular transport particle in neurons (27 29). In particular, there are no good candidates for the critical adaptor and anchoring proteins that would link mrnps to transport and targets, respectively. RNA granules RNA granules were first defined in oligodendrocytes as structures containing the mrna-encoding myelin basic protein and translational components, including ribosomal mrna and ribosomes (30,31) that are important for the transport of myelin basic mrna to the myelinating regions of these cells. It is important to stress that translation is also repressed in these structures, despite the presence of ribosomes. In neurons, Kosik and colleagues described a structure identified in the electron microscopy as amorphous collections of ribosomes that contained Staufen I (2,32). The presence of aggregates of ribosomes in RNA granules implies that mrnas in RNA granules are translationally repressed at the stage of elongation, in contrast to mrna in transport particles that are translationally repressed at the stage of initiation. We feel that these fundamentally distinct types of transport structures should be distinguished by different names. As recent reviews have defined neuronal RNA granules by the presence of both large and small subunits of ribosomes, distinguishing them from other RNA structures (33), we think keeping this name for this structure is conservative. The RNA transport particles that do not contain ribosomes should not be called RNA granules, but RNA particles to distinguish these two mechanisms. The RNA granules have been extensively studied in neurons, and two proteomic studies of these granules will be discussed below. Proteomics of RNA Granules The RNA granules have been purified and characterized using proteomics to determine the underlying protein components of these granules. There were two different purifications, one from adult brain purified based on the association of the granule with kinesin heavy chain KIF5 (34), and the other based solely on subcellular fractionation from embryonic brain (35). Despite the differences in the protocols used, there were many shared components of these granules, suggesting that some of the core proteins of the RNA granule have been identified. There were also some major differences in these granules, in particular the motors coupled to transport (kinesin for the adult brain and dynein for the embryonic brain). Whether these represent developmental differences or are due to distinct types of RNA granules in neurons is not yet clear. One striking finding from both studies was the abundance of hnrnps and other nuclear proteins in the granule. While hnrnp-a2 (36) and hnrnp I (37) had been implicated in RNA transport previously, most models of RNA processing would suggest that hnrnps are only transiently associated with cytoplasmic mrnas and then are quickly cycled back to the nucleus (38). However, even proteins largely nuclear in localization, such as hnrnp C and hnrnp U, were found on RNA granules. Another interesting finding from the proteomics is the presence of several DEAD box proteins. DEAD box 1, 3 and 5 were found in both sets, while DEAD box 6 (homologue of yeast Dhh1P, Rck54 and Drosophila Me31b) and DEAD box 9 were also found in the embryonic RNA granule. DEAD box proteins may be important in the organization of RNA granules as exemplified by the role of VASA in the formation of polar granules in Drosophila oocytes (39). Dhh1P has been proposed to play an organizing role in the formation of P bodies (5). The presence of Dhh1P in both RNA granules and P bodies may suggest some organizational similarities; however, ribosomes are not found in P bodies and decapping enzymes have not been found in RNA granules. One novel protein was isolated in both proteomic screens, CGI-99. This protein is highly conserved over evolution, but does not appear to have a known RNA-binding domain, and thus is a good candidate for being one of the elusive adaptor proteins critical for the formation of RNA transport structures. Elucidation of its function may lead to a better understanding of RNA granules. It is not clear whether RNA granules are a homogenous or heterogeneous structure. In the work on the kinesinassociated granule, most identified proteins appeared to co-localize in a single structure. In contrast, work on the embryonic RNA granule found at least two distinct types of puncta in neurons, one enriched in DEAD Box 3 and the other containing CGI-99 (35). Interestingly, neither of those puncta showed strong co-localization with Staufen 2. Thus, at least during neuronal development, there appears to be distinct subtypes of neuronal granules. In oligodendrocytes, three distinct ribosome-containing puncta have been observed at myelinating sites, one containing Staufen 1, one Staufen 2 and a third containing hnrnp-a2 and the myelin basic protein mrna (40). Nuclear Origin for RNA Transport The presence of hnrnps and other mainly nuclear proteins in both RNA granules and transport particles suggest that formation of the RNA granule begins in the nucleus (41). Indeed, studies examining the assembly of transported mrnas demonstrate initial association of mrnas with RNA-binding proteins involved in transport occurred in the nucleus (42,43). Moreover, proteins that associate in the nucleus at exon junctions, such as Barentz, are also found 1584 Traffic 2006; 7:

5 RNA Trafficking in Neurons in putative transport particles (44). In vertebrates, proteins associated with the exon junction are used to check for truncated or aberrantly spliced transcripts through nonsense-mediated decay (45), while in Drosophila, these proteins have also been associated with transport (46). The presence of exon-junction proteins on transport particles may suggest that transport is initiated before the transcript is assessed during a pioneer round of translation for non-sense-mediated decay because active translation is thought to remove the EJC from mrnas (45). The RNA granules, however, do not appear to contain exon-junction proteins as assessed by proteomics, consistent with transport being initiated after translation initiation. It should be noted that if nuclear assembly is important for the formation of transport particles and/or RNA granules, then studies using transfection of RNAs may measure a different type of transport than ones using transfection of plasmids that are first transcribed in the nucleus. Transport and Translational Repression The transport of mrna is tightly coupled to the repression of translation. If RNA transport particles are used, then repression will be at the step of initiation, as mrnas are not associated with ribosomes. However, if mrnas are stored in RNA granules after formation of polysomes, then repression will be at the level of elongation. We will discuss a number of models for regulating translational repression and their relationship with transport. Repression by eif4e-binding proteins The eif4e-binding proteins repress translation by preventing eif4e binding to the adaptor eif4g and thus to the ribosome (47). The classic eif4e-binding protein is eif4e- BP whose binding to eif4e is regulated by phosphorylation. However, while local translational regulation downstream of eif4e-bp phosphorylation is important in local translation in dendrites (48), there is no data proposing a role for eif4e-bp in repression of mrnas during transport nor data suggesting localization of eif4e-bp to either RNA transport particles or RNA granules. However, other binding partners of eif4e do play this role, probably as they can restrict eif4e binding to eif4g on specific mrnas through association with both eif4e and an RNA-binding protein. In Xenopus, Maskin binds to eif4e and to cytoplasmic poly(a)-binding protein (CPEB) blocking translation until the appropriate stimulus arrives (49). It is not clear if mammalian Maskin plays the same role because it lacks the canonical eif4e-binding domain; however, other mammalian proteins have been recently discovered that bind both CPEB and eif4e, suggesting this mechanism may be important in repressing transport of CPEB-regulated mrnas (50). The CPEB is not only involved in repression but may also play a direct role in transport because loss of CPEB leads to a specific reduction in the transport of mrnas containing a binding site for CPEB (51). In Drosophila, cup is an eif4e-binding protein that binds directly to a number of RNA-binding proteins like Bruno and is important in repression of translation during transport in the oocytes (52,53). In vertebrates, an eif4ebinding protein, eif4e-t, that shows some homology to Drosophila cup binds to eif4e and appears to play a role in repressing translation and moving mrnas to P bodies (54). Interestingly, in another Drosophila transported mrna, eif4e is replaced by an alternative isoform, eif4ehp, that binds to a distinct RNA-binding protein (55). This form of repression is linked specifically to RNA transport particles whereas RNA granules appear to lack eif4e (2,35). Represssion by deadenylation The mrnas can be translationally repressed by removing the poly(a) tail and thus the presence of poly(a)-binding protein, which, similar to eif4e, binds to eif4g and is important for translation initiation. The mrnas are then derepressed by cytoplasmic poly(a) that is under the control of CPEB. This mechanism has been shown to be important, particularly in oocytes, but also in neurons, where stimulation leads to poly(a) addition (56). The current model is that mrna-binding proteins, such as Pumilio, bind to sequences in the 3 0 UTR and recruits other factors to form an RNA protein complex that causes deadenylation of mrna and translational repression (57). Repression by mirnas The mirnas may be the most prevalent mechanism for inhibiting translation in cells, as there are a large number of mirnas, especially in the nervous system, and each mirna can repress multiple messages (18). The mirna precursors are exported from the nucleus and then processed by the argonaute complex and stored in the RISC complex. Two recent studies have implicated mirnas in the repression of mrnas transported to dendrites, one from Drosophila for CAMKII mrna (58) and another for LIMK mrna during neurite development in mammals (59). Thus, mrnas localized in dendrites can be repressed by mirnas. A major question left unanswered by these studies was whether the binding of mirnas is involved in repression during or after transport. It is likely that both mechanisms can operate depending on specific additional regulators of particular mrnas. Interestingly, degradation of an important component of the RISC complex in Drosophila led to increased translation of CAMKII at the correct location, suggesting that transport was independent of the mirna, but repression at the target depends on mirnas (58). It is not clear whether mirnas are already associated with the transported mrnas or bind to mrnps formed after transport. Repression by binding to other RNA-binding proteins A number of mrna-binding proteins have been shown to repress translation (both in vitro and in vivo) and are associated with transported mrnas, although their mechanisms of action are not yet well defined. Traffic 2006; 7:

6 Sossin and DesGroseillers RNA granule 105 (RNG105), also known as Caprin-1, is an RNA-binding protein found as puncta in dendrites of neurons and co-localizes with Staufen and ribosomes, suggesting RNG105 is a component of mrna granules (3). Consistent with this, RNG105 was detected in the proteomic screen of RNA granules from developing brain (35). Overexpression of RNG105 represses translation both in rabbit reticulocyte lysates and in cell cultures (3). Inhibition does not seem to be RNA sequence specific. This protein was also identified as a protein interacting with the receptor for activated C kinase (RACK), although at the time is was misnamed as a glycosyl-phosphatidylinositol-linked protein in the database (60). RACK is a protein that associates with the large ribosome and may regulate subunit joining through eif6 (61) and was also identified in RNA granules by proteomics (35). Surprisingly, RNG105 also co-localized with eif4e in dendrites (3). It was not clear if the puncta containing eif4e and RNG105 were the same puncta as those containing RNG105 and ribosomes. The zip-code-binding proteins (ZBP1-3) were identified as an RNA-binding protein that bound to the region of the b-actin 3 0 UTR that is required for transport (62) but binds many other mrnas as well. Interestingly, ZBP appears to be a broadly conserved localization factor because Vera, a homologue of ZBP in Xenopus, is important for transport of Vg1 mrna to the vegetal pole (63). The ZBP was found in one of the proteomics studies, suggesting that it can be associated with RNA granules but is also probably present in RNA particles as well. ZBP has been shown to repress translational initiation of the b-actin mrna (64) and to be required for its transport in neurons (65). The hnrnp-a2 was initially implicated in transport of myelin basic protein in oligodendrocytes based on its binding to the critical region required for transport (66). It has recently been shown to be important for transport of mrnas encoding an hnrnp-a2-binding site in neuronal dendrites as well (36). The hnrnp-a2 appears to repress translation through binding to hnrnp-e1 (67). FMRP is one of the most heavily studied mrna repressor molecules. There is strong biochemical and genetic evidence that it regulates local translation of transported mrnas in neurons (68). In particular, FMRP is strongly implicated in the regulation of local translation in dendrites. Long-term synaptic depression (LTD) induced by the activation of metabotropic glutamate receptors requires local dendritic synthesis. In Fragile X knock out animals, this form of LTD is enhanced and no longer requires protein synthesis, suggesting that de-repression of FMRP-repressed mrnas is critical for this form of local plasticity (69). However, exactly which messages are regulated and the mechanism of repression used is still unclear (70), although a fraction of phosphorylated FMRP was shown to be associated with apparently stalled polysomes (71). Stalled polysomes A model for the repression of translation in RNA granules containing polysomes is that translation is stalled. The mechanism underlying this is not known, although there are several examples where translation of mrnas is repressed while associated with polysomes. Repression of nanos in Drosophila oocytes occurs while the nanos mrna is associated with polysomes (72), although in this case, translation is not stalled but somehow the protein is degraded as it is translated. An element in the 3 0 UTR is required for the repression through the RNA-binding proteins Smaug and Glo, a homologue of hnrnp H/K. These hnrnps were found in the D proteomics of RNA granules. Repression of oskar translation in Drosophila may also be at the polysome phase as the mrna is found in polysomes and the Nac alpha protein, a chaperone, is required for efficient repression (73). Both Nac alpha and the TCP chaperone were found in the proteomics of RNA granules from neurons as well (35), suggesting that nascent proteins may also be present on these structures. Transport and De-repression of Translation While mrnas are translationally repressed during transport, translation needs to be reactivated once localized. The molecular mechanisms that regulate this essential step are still unclear. However, recent studies begin to shed light on these mechanisms. In particular, neuronal activation was shown to cause the translocation of mrna from RNA granules to polysomes. In neurons, RNG105 dissociates from RNA granules in dendrites following treatment of the cells with BDNF and this is correlated with the translation of a reporter construct (3). Similarly, mrna in Staufen-containing granules translocate from granules to polysomes after synaptic activation (2). The molecular signal that triggers this translocation is not known, although phosphorylation/de-phosphorylation steps of critical RNA-binding proteins would be logical. In fibroblasts, translational repression exerted by ZBP on the b-actin mrna is released through Src-dependent phosphorylation of ZBP whose affinity for b-actin mrna decreases (64). Similarly, phosphorylation of FMRP regulates its ability to repress translation. While overexpression of FMRP leads to an increase in stalled polysomes, nonphosphorylated FMRP loses this ability, suggesting that its dephosphorylation may lead to the release of polysomes from the stalled state (71). Staufen, the protein probably most associated with mrna trafficking in neurons (74) may also be part of the mechanism that regulates translational de-repression. In Drosophila, in addition to its role in the transport of oskar mrna to the posterior pole, genetic and molecular studies revealed that Staufen is also necessary for the translational de-repression of oskar mrna once localized (75). Consistently, in mammalian cells, Stau1 was shown to de-repress 1586 Traffic 2006; 7:

7 RNA Trafficking in Neurons translation of some mrna when bound to their 5 0 UTR (76). Stau1 has no effect on the translation of most of the RNA because it must bind the mrna 5 0 UTR via recognition of a Staufen-binding site to enhance translation. Staufen is also known to play other roles in cells. First, both Staufen 1 and Staufen 2 were shown to transit through the nucleus (77,78). It was suggested that they might be involved in selection of transported mrna in the nucleus and/or their export to the cytoplasm. Second, Staufen 1 regulates mrna stability of a population that contains a Staufen-binding site in their 3 0 UTR (79). Binding of Staufen 1 downstream of an endogenous stop codon causes recruitment of the non-sense-mediated RNA decay factor UpF1 and degradation of the bound mrna. Therefore, Staufen 1 has the ability to regulate RNA transport at different steps, including RNA selection and export from the nucleus, cytoplasmic transport on the cytoskeleton, de-repression of translation once localized and RNA degradation to terminate the signal. Whether Staufen plays all these successive roles in the transport and translation of a single RNP is still unclear, as well as the regulation of all these functions in response to cell needs. Different RNA Transport Mechanisms for Different Functions in Neurons It is unlikely that all transport in neurons uses the same mechanism. After all, during development, mrnas are sorted into both axons and dendrites, and there are many differential functions of mrnas once transported that might benefit from distinct modes of transport. After transport, it is not clear what the fate of mrnas is in neurons. Are they anchored initially awaiting a stimulus for de-repression, does the target site induce de-repression itself or are they constantly on the move until an activating signal is generated? After de-repression, are the mrnas degraded, stored in P bodies or recycled into another transport particle or RNA granule? These dynamics of RNA trafficking are uncharacterized but critical for the understanding of local translation in neurons. Gene expression is required for long-term synaptic changes and long-term memory. However, it has not yet been shown that transport of these newly synthesized mrnas is required for long-term changes. For instance, it is possible that these mrnas are translated in the cell body and the proteins sent out to synapses for their function in the formation of long-term synaptic changes. Even if RNA-binding proteins are implicated in memory formation, such as Staufen and Pumilio in Drosophila (80), it is not necessarily due to the lack of localization of newly synthesized mrnas. For example, removing the 3 0 UTR from CAMKII in a knock-in mouse impaired the formation of long-term memory but also reduced basal levels of CAMKII in dendrites by about 90% (81). Thus, the deficit in long-term memory was probably due to the lack of constitutive transport of CAMKII, changing the levels of proteins present that are required to induce plasticity. Additionally, transport of mrnas is critical for development. For example, sirna of Staufen II greatly decreased the number of synapses, presumably due to the lack of transport of mrnas needed in the dendrite for proper growth (82). Blocking ZBP binding to mrnas reduced growth cone motility (65). Mutations in Fragile X also lead to abnormal dendritic development that may be the cause for the mental retardation associated with this phenotype (68). It will be difficult to disassociate the need for mrna transport to properly set up the compartmentalization of dendrites with their specific proteins and a possible need for mrna transport to localize newly synthesized mrnas for learning and memory unless different transport mechanisms are used for these different pools. This may be the case, for instance, Arc mrna is not present before a learning stimulus but is transcribed quickly and then transported to active synapses (83). This stimulus does not lead to the relocalization of mrnas that are already present in dendrites to activated synapses (83). This could be due to a different transport mechanism or due to the fact that the majority of mrna for the constitutively transported mrnas are no longer in transport particles or RNA granules and thus do not relocalize. Summary The transport of RNA in neurons is a critical but complex feature. In this review, we have highlighted recent advances and also raised a number of important issues for future research. Are different transport structures in neurons used for distinct functions? What are the dynamics of RNPs in neurons and when and where do they cycle through different RNA structures? In the next years, answers to these questions should help us understand this fascinating process. References 1. Brengues M, Teixeira D, Parker R. Movement of eukaryotic mrnas between polysomes and cytoplasmic processing bodies. Science 2005;310: Krichevsky AM, Kosik KS. Neuronal RNA granules: a link between RNA localization and stimulation-dependent translation. Neuron 2001;32: Shiina N, Shinkura K, Tokunaga M. A novel RNA-binding protein in neuronal RNA granules: regulatory machinery for local translation. J Neurosci 2005;25: Sheth U, Parker R. 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