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The nuclear nurture and cytoplasmic nature of localized mRNPs
Seminars in Cell & Developmental Biology 18 (2007) 186–193
Review
The nuclear nurture and cytoplasmic nature of localized mRNPs Corinna Giorgi, Melissa J. Moore ∗ Department of Biochemistry, Howard Hughes Medical Institute, Brandeis University, Waltham, MA 02454, USA Available online 23 January 2007
Abstract From yeast to mammals, evidence has emerged in recent years highlighting the essential role played by the nuclear “history” of a messenger RNA in determining its cytoplasmic fate. mRNA localization, translation and stability in the cytoplasm are often pre-destined in the nucleus, and directed by the composition and architecture of nuclear assembled mRNA–protein complexes. In this review we focus on nuclear-acquired RNA-binding proteins and complexes that participate in determining the journey of localized mRNAs. © 2007 Published by Elsevier Ltd. Keywords: mRNA localization; Exon junction complex; Nuclear mRNA processing
Contents 1. 2.
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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The exon junction complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. The EJC, translation and NMD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. The EJC and mRNA localization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Other nuclear-acquired mRNA binding proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. ZBP1 proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.1. Ash1 mRNA and associated factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. HnRNP and SR proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.1. HnRNP A2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.2. Squid and Hrp48 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Neuronal transport granules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction A perennial debate among psychologists concerns the varying degrees to which Nature and Nurture contribute to human behavior and destiny. Nature is generally defined as an individual’s innate qualities (i.e., genetic makeup), while Nurture refers to personal experiences (i.e., one’s emotional and physical environment, particularly during prenatal development and childhood). Although historically this debate has swung widely ∗
Corresponding author. Tel.: +1 781 736 2359; fax: +1 781 736 2337. E-mail address: [email protected] (M.J. Moore).
1084-9521/$ – see front matter © 2007 Published by Elsevier Ltd. doi:10.1016/j.semcdb.2007.01.002
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between the two extremes, a more modern view divides human qualities into three broad categories: exclusively genetic (e.g., blood type and eye color), exclusively environmental (e.g., languages spoken and cultural beliefs) and interactional (e.g., height, weight and almost all psychological traits such as IQ). Recent advances in our understanding of gene expression now make it clear that parallel forces are at work at the molecular level determining the behavior and destiny of eukaryotic mRNAs. Here Nature can be defined as the primary sequence of the nascent transcript and Nurture as the nuclear environment in which it is processed and assembled with trans-acting factors into a mature and exportable mRNP. In contrast to the three broad categories of human qualities, however, it now seems that
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virtually all traits and behaviors of eukaryotic mRNAs are interactional. For example, even the most basic property of what protein is encoded by an mRNA is not entirely attributable to the primary sequence of the original transcript. Rather it depends on its splicing and polyadenylation patterns, which in turn are functions of both pre-mRNA sequence and the unique set of trans-acting factors encountered during assembly of the processing machinery. This set of trans-acting factors depends on cell type and cellular environment (e.g., signal transduction pathways) as well as pre-mRNA transcription rate and the intranuclear location of the gene. In this review, we will focus on how a history of nurturing in the nucleus affects subsequent mRNA behavior and destiny in the cytoplasm. Behaviors exhibited by individual mRNAs in the cytoplasm include targeted localization to one or more specific subcellular compartments, variations in where and when the translation apparatus is engaged and differential half-lives. As is the nuclear process of pre-mRNA splicing, all of these cytoplasmic behaviors are interactional; that is, they are a function of both the primary mRNA sequence and the trans-acting factor environment. Emergent themes are (1) that much of what happens to an mRNA in the cytoplasm, including its localization, is pre-destined by trans-acting factors acquired in the nucleus and (2) the intimate association between mRNA localization and translational repression. 2. The exon junction complex Perhaps the best known example of how the nuclear history of an mRNA can affect its cytoplasmic destiny is provided by the exon junction complex (EJC). The EJC is a set of proteins
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deposited on mRNAs as a consequence of pre-mRNA splicing. This deposition occurs ∼20 nts upstream of the site of intron excision and has no apparent mRNA sequence requirement [1]. Although not yet formally proven, it is generally assumed that an EJC is deposited upstream of every exon junction on all spliced mRNAs. Once bound, EJCs travel with the mRNA to the cytoplasm where most are ultimately removed as a consequence of the first or “pioneer” round of translation [2,3]. Prior to their displacement, however, they can act as effectors of almost every aspect of mRNA metabolism including subcellular mRNA localization, mRNA translational efficiency and ultimately mRNA decay (Fig. 1) [4–6]. Structurally, the EJC consists of a stably bound, heterotetrameric core that serves as a binding platform for other more transiently associating factors [7]. At the heart of this core is eIF4AIII, the main RNA anchoring protein [8]. A member of the DEAD-box family of ATP-dependent RNA remodeling proteins, eIF4AIII exhibits ATP-dependent RNA binding activity. During EJC assembly, eIF4AIII presumably becomes locked on the RNA when the Y14:MAGOH heterodimer joins and inhibits its ability to hydrolyze ATP. A fourth core component, MLN51, serves to increase eIF4AIII’s inherent affinity for RNA [9]. Two recent crystal structures of the eIF4AIII:Y14:MAGOH:MLN51 heterotetramer bound to RNA are notable in that they represent the first structures of any DEAD-box protein in complex with multiple binding partners which modulate its activity [10,11]. The list of more peripherally associating EJC proteins includes numerous factors involved in pre-mRNA splicing, mRNA export, nonsense-mediated mRNA decay (NMD) and programmed cell death. However, only a subset of these factors has been shown to remain associated with spliced mRNAs in
Fig. 1. Roles played by the EJC in cytoplasmic metabolism of mRNAs. The EJC has been implicated in aiding mRNA localization, modulating translational efficiency and allowing detection of target mRNAs for the nonsense-mediated decay pathway. P-bodies are cytoplasmic sites of mRNA turnover and mRNA storage.
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the cytoplasm. This subset consists of the splicing factor RNPS1 and the NMD factors Upf2 and Upf3b [7]. RNPS1 was originally characterized as a splicing co-activator capable of increasing the efficiency of pre-mRNA splicing in vitro [12]. It is also a component of the trimeric ASAP complex implicated in the induction of apoptosis [13]. Upf2 and Upf3b, along with Upf1, are central components of the nonsense-mediated mRNA decay pathway (see below). Whereas RNPS1 and Upf3b originally associate with the EJC core in the nucleus, Upf2, a perinuclear protein, presumably joins the complex soon after the spliced mRNA enters the cytoplasm [6].
and Upf2 and Upf3b bound to a downstream EJC [17]. This triggering of NMD can be replicated independent of splicing: Upf and certain EJC factors, if artificially tethered to the 3 UTR of a reporter mRNA, can induce decay of the transcript [19]. In contrast, tethering of the same proteins inside the open reading frame (ORF) increases translational yield, replicating the effects of ORF-bound EJCs in polysome recruitment as above. At present, it is unknown how these disparate positional effects, one enhancing translation and the other leading to mRNA degradation, are mechanistically related. Nonetheless, given that they involve an identical set of proteins, it is tempting to speculate that a connection does exist [6,16,20].
2.1. The EJC, translation and NMD 2.2. The EJC and mRNA localization Initial observations linking pre-mRNA splicing to cytoplasmic mRNA metabolism came from studies comparing the translational efficiency or decay of spliced versus cDNA-derived transcripts. In Xenopus oocytes, more protein is produced in the cytoplasm upon nuclear injection of an intron-containing pre-mRNA than upon injection of the corresponding cDNA transcript ([14] and references therein). This effect is readily observable in mammalian cells as well, where intron-containing and cDNA versions of otherwise identical genes yield significantly different amounts of protein per molecule of cytoplasmic mRNA [5,15]. This influence of splicing on translational yield has been directly linked to EJC deposition, and sedimentation analysis indicated that a greater proportion of spliced mRNAs are polysome-associated than unspliced mRNAs [16]. Though the exact mechanism of how the enhanced polysome association is achieved has yet to be elucidated, one model is that EJCs aid in recruiting translation initiation factors to promote the pioneer round of translation. Another possibility is that, prior to their removal by the pioneer round, EJCs assist in delivering newly exported mRNAs to regions of the cytoplasm (e.g., the cytoskeleton) enriched in ribosomes. Regardless of the mechanism, one potential advantage of such enhanced translation of EJC-carrying mRNAs is that it allows for preferential translation of newly transcribed messages, thus decreasing any delay time between transcriptional activation and protein expression. Whereas EJCs in the coding region can enhance gene expression by increasing translational yield, EJC deposition in the 3 -UTR has the opposite effect of triggering mRNA decay via the NMD pathway. NMD is generally viewed as a protective mechanism allowing cells to eliminate aberrant mRNAs containing pre-mature termination codons (e.g., those produced from mutant alleles or by improper pre-mRNA processing), which might otherwise direct the synthesis of truncated and potentially dominant-negative proteins [17]. Another, and likely as important, function of the NMD pathway is to regulate expression of natural NMD targets. Such natural targets include mRNAs containing upstream open reading frames (uORFs) and those produced from transcripts harboring introns in the 3 -UTR [17,18]. In mammalian cells, a major trigger for the NMD process is a stop codon located >50 nts upstream of at least one exon–exon junction. A proposed model for this type of NMD entails interaction between Upf1 bound to a ribosome stalled at the stop codon
A third known function for the EJC is in localization of oskar mRNA in the developing Drosophila oocyte. Restricted translation of oskar at the posterior pole is essential for germ line and abdomen formation in the future embryo [21]. Oskar mRNA is initially synthesized in the adjacent nurse cell nuclei and then transported into the oocyte through ring canals connecting the nurse cell and oocyte cytoplasms. Transport within the oocyte occurs along microtubules in a plus-ended direction mediated by the motor protein kinesin [22]. Extensive genetic analyses have identified a plethora of trans-acting factors required for translational repression, transport, anchoring and subsequent translational activation of oskar mRNA at the posterior pole. Among the factors required for transport are all four of the EJC core proteins: eIF4AIII, Barentsz, Mago Nashi and Tsunagi (the Drosophila orthologs of MLN51, Magoh and Y14, respectively) [4,23]. All four of these proteins initially accumulate with oskar mRNA at the posterior pole. Although the EJC core factors are essential for proper oskar mRNA localization, it was not initially thought that pre-mRNA splicing had any involvement. This was because chimera experiments had shown that the 3 -UTR of oskar mRNA, which contains no introns, is sufficient to direct posterior pole localization of an intronless reporter when expressed in an otherwise wild type egg chamber [24]. However, this is not the case in egg chambers lacking endogenous oskar mRNA [25]. Under these conditions, proper localization of oskar mRNA expressed from an exogenous transgene requires that the transgene contain at least one intron. This result clearly demonstrates that splicing is required for oskar mRNA localization. Subsequent analysis revealed that the oskar 3 -UTR is capable of dimerization via binding of the trans-acting factor Bruno to its Bruno response elements (BREs) [26]. This nicely accounts for the previous finding that an intronless oskar chimera can be properly localized in the presence of endogenous oskar mRNA, as the chimera could heterodimerize and thereby be transported piggyback style with the endogenous EJC-carrying transcripts. The requirement of EJC deposition for proper oskar expression highlights an emerging connection between mRNA localization and translational repression. Because all three introns in the pre-mRNA lie within the coding region, all oskar EJCs are subject to removal by the pioneer round of translation. Thus, in order to maintain the EJC association necessary
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for its localization, oskar mRNA must not undergo translation prior to arrival at its target destination. While oskar is the clearest example to date of an mRNA requiring translational silencing for localization, more and more examples are emerging of links between these two processes (see below). Thus, oskar may exemplify the generality that localized mRNAs are translationally repressed prior to and during transport. Whereas no direct evidence yet exists for involvement of the EJC in localizing other mRNAs in other organisms or cell types, circumstantial evidence may be pointing in that direction. For example, Magoh, Y14 and MLN51 all localize to the dendrites of mammalian hippocampal neurons [27–29]. These highly polarized cells rely on local translation of dendritically targeted mRNAs to modulate the activity of individual synapses. Within dendrites, MLN51 associates with the known mRNA transport factor Staufen 1 and the abundant transported small RNA, BC1, which has been implicated in the translational regulation of localized mRNAs. Furthermore, data from our own laboratory indicate that eIF4AIII exists in dendritic mRNA localization particles and might be involved in regulating local protein expression at synapses (C.G. and M.J.M., unpublished data). Thus it seems that we have only begun to scratch the surface of the EJC’s involvement in cytoplasmic mRNA metabolism. 3. Other nuclear-acquired mRNA binding proteins Although the EJC has garnered much attention in recent years, it is but one example of an ever-expanding list of nuclearacquired factors that impact cytoplasmic mRNA metabolism [30]. The remainder of this article focuses specifically on those factors whose function can be linked to proper expression of localized mRNAs. 3.1. ZBP1 proteins One of the best examples of nuclear assembly of a transport-competent mRNP involves association of the zipcodebinding-proteins 1 and 2 (ZBP1/2) with -actin mRNA. Members of the ZBP family contain RRM and KH RNAbinding domains and mediate cytoplasmic mRNA localization in a variety organisms and cell types. In chick embryo fibroblasts, neuronal growth cones and rat hippocampal neurons, proper cell structure and motility require asymmetric localization of -actin mRNA driven by ZBP1 (reviewed in [31]). ZBP1 recognizes a 54 nucleotide localization element, the zipcode, in the -actin 3 -UTR. In fibroblasts, mutations in this zipcode or treatment with zipcode-antisense oligonucleotides lead to delocalization of -actin mRNA and alteration of cell motility [32,33]. In mammalian neurons, ZBP1 is required for the dendritic targeting of -actin mRNA, and altered ZBP1 levels affect the density of dendritic filopodia, actin-rich dendritic protusions involved in synaptogenesis [34,35]. ZBP1 is predominantly cytoplasmic. Nonetheless it contains Rev-like nuclear export and nuclear localization sequences, and shuttles between the nucleus and cytoplasm utilizing the Crm1 export pathway [32]. Using a combination of in situ hybridization and immunofluorescence in chicken fibroblasts, Oleynikov
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and Singer demonstrated that ZBP1 associates with -actin mRNA at the site of transcription. Furthermore, impeding ZBP1 nuclear export with Leptomycin B affected cytoplasmic -actin mRNA transport [32]. A second nucleo-cytoplasmic shuttling protein, ZBP2, also binds the -actin zipcode element. Although ZBP2 is predominantly nuclear, a small portion does co-localize with cytoplasmic -actin mRNA in both chick fibroblasts and neurons, suggesting that ZBP2 travels out of the nucleus aboard the mRNA. A direct role of nuclear-acquired ZBP2 in mediating cytoplasmic targeting of -actin mRNA was suggested by the finding that overexpression of a truncated ZBP2 suppresses -actin mRNA localization in fibroblasts and neurons [36]. ZBP2-related proteins include the splicing factor FBP2/KSRP in humans, MARTA1 in rodents, and VgRBP71 in Xenopus. Like ZBP2, both MARTA1 and VgRBP71 are predominantly nuclear. Nonetheless, they have been implicated in cytoplasmic mRNA localization of Map2 mRNA in mammalian dendrites and of several localized mRNAs in Xenopus oocytes, respectively. This suggests a conserved role for nuclearacquired ZBP2-like proteins in cytoplasmic mRNA localization [37,38]. A link between nuclear ZBP acquisition and cytoplasmic mRNA localization was also recently reported in Xenopus oogenesis. Vg1 and VegT are two maternal mRNAs that localize at the vegetal pole and have been implicated in mesoderm and endoderm specification of the embryo. Their vegetal pole localization is directed in part by the trans-acting factors Vg1RBP60/hnRNPI and VERA/Vg1RBP, the Xenopus homologue of ZBP1. These factors bind to 3 -UTR localization elements (LEs) in the transported mRNAs and mutations in the LEs that disrupt protein binding lead to mislocalization of the transcripts [39]. Immunoprecipitation of tagged Vg1RBP60/hnRNPI and VERA/Vg1RBP revealed that both proteins associate with Vg1 and VegT mRNAs in the nucleus as well as the cytoplasm [40]. A recent report by the Singer lab [41] demonstrates that in addition to directing -actin mRNA to the leading edge of fibroblasts, ZBP1 is also involved in repressing its translation both in vitro and in neuroblastoma cells. ZBP1 therefore seems to couple transport with translational repression of -actin mRNA. A combination of in vitro and in vivo approaches also provided evidence for how the translational repression can be reversed once the mRNA is localized. The suggested mechanism involves phosphorylation of ZBP1 by the tyrosine kinase Src resulting in diminished affinity for the bound mRNA and its consequent emancipation from translational repression. SRC involvement in regulating asymmetric -actin mRNA expression was also reported in a recent study linking localization of both species to Ca2+ -dependent growth cone guidance in Xenopus laevis spinal neurons [42]. 3.1.1. Ash1 mRNA and associated factors One of the best-understood examples of active mRNA transport (i.e., requiring molecular motors) is provided by S. cerevisiae ASH1 mRNA [43]. During cell division, ASH1 mRNA migrates from the mother cell to the bud tip where its localized
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expression is key for suppressing mating type switching in the daughter cell. ASH1 mRNA localization requires direct association of the RNA-binding protein She2, a nucleocytoplasmic shuttling protein, with specific mRNA sequence elements. She2 then recruits She3, which in turn interacts with She1, a type V myosin. In addition to its key role in localization, She2 binding to three sites on ASH1 mRNA coding region is required for translational repression of the mRNA during transport. Mutant mRNAs in which She2 binding sites are moved to the 3 UTR retain the ability to localize but are translationally derepressed during transport [44]. The nuclear connection in this system comes in the form of Loc1, a nuclear restricted protein required for the initial recruitment of She2 to ASH1 mRNA [45]. 3.2. HnRNP and SR proteins Heterogeneous nuclear ribonucleoproteins (hnRNPs) comprise a diverse set of proteins originally identified by their association with hnRNA. Characteristic of this class is the presence of one or more RNA binding domains, including RRMs, RGG and KH domains [46]. Another major class of RNAbinding factors is the SR proteins, which, in addition to a N-terminal RRM, typically have a C-terminal domain rich in RS (arginine-serine) repeats. SR proteins are subject to dynamic methylation and/or phosphorylation within the RGG and RS regions. Such modifications, which can be compartment limited, can alter the RNA binding properties of the modified protein [47–52]. HnRNPs and SR proteins can also be subclassified according to whether or not they shuttle in and out of the nucleus [53,54]. It is the shuttling class that is relevant for the discussion below. Generally these factors associate with mRNA in the nucleus, many co-transcriptionally, and, like the EJC, accompany mRNAs to the cytoplasm where they modulate mRNA localization, translational efficiency and mRNA stability. Many also have important and distinct functions in the nucleus in addition to their cytoplasmic roles [46,47,55]. 3.2.1. HnRNP A2 One of the best understood hnRNPs involved in mRNA localization is hnRNP A2 (a.k.a. hnRNP A/B). HnRNP A2 is required for the localization of myelin basic protein mRNA (MBP mRNA) to myelin-forming processes in mammalian oligodendrocytes [56]. Necessary and sufficient is the 11 nucleotide A2 response element (A2RE) located in the MBP mRNA 3 UTR. Introduction of this element into a non-localized GFP reporter is capable of driving its extrasomatic localization when microinjected in oligodendrocytes [57]. Further, when the A2RE contains mutations interfering with hnRNP A2 binding, the mutant mRNA fails to localize properly. Finally, microinjection of either antibodies or antisense oligonucleotides targeting hnRNP A2 adversely affects trafficking of microinjected A2RE RNAs ([58] and references therein). A2RE-like sequences are also found in several other mRNAs, including three HIV transcripts and the dendritically localized CaMKII␣, Arc, Neurogranin and MAP2 mRNAs, although only a subset of these elements has been tested in an RNA localization assay [57–60].
Beyond its role in mRNA localization, recent data also suggest a possible involvement of hnRNP A2 in modulating translation of the cargo mRNA. Within A2RE-mRNP transport granules in oligodendrocytes, hnRNP A2 interacts and co-localizes with hnRNP E, a predominantly cytoplasmic translational repressor. Functional assays revealed that hnRNP A2 serves to recruit hnRNP E to A2RE-containing reporter mRNAs in vivo, resulting in translational inhibition [61]. 3.2.2. Squid and Hrp48 In Drosophila, the hnRNPs Squid and Hrp48 (closely related to mammalian hnRNP A1 and A2, respectively) are involved in cytoplasmic mRNA localization during oogenesis (reviewed in [21]). Squid binds directly to the 3 -UTR of gurken mRNA and this association is required for proper localization of gurken to the dorso-anterior corner of the oocyte [62]. Hrp48 binding to oskar mRNA in both the 5 and 3 portions of the transcript is essential for the proper delivery of oskar to the posterior pole [63]. In an interesting twist, recent work indicates that localization of oskar mRNA to the posterior pole also requires Squid, and, conversely, Hrp48 participates in localization of gurken mRNA to the dorso-anterior corner [64,65]. This suggests that both of these proteins act as transport competency factors, but neither determines the ultimate destination of the mRNA within the oocyte. Drosophila Hrp48 and Squid also participate in repressing translation of oskar and gurken mRNAs, respectively [66], offering another example of the tight coupling between cytoplasmic targeting and translational repression of localized mRNA. While Squid seems to bind gurken mRNA in the nucleus [62], it is at present unclear where Hrp48 first associates with oskar mRNA. In contrast to its mammalian homolog (hnRNP A2), Hrp48 is predominantly cytoplasmic [67]. Nonetheless, a small fraction does exists in the nucleus where it has been shown to participate in alternative splicing of nascent mRNAs ([68] and references therein), so Hrp48 could certainly be a nuclearacquired mRNP factor. 3.3. Neuronal transport granules HnRNPs have also been found in neuronal transport granules. These large macromolecular assemblies are found in dendrites and include dendritically targeted mRNAs plus numerous trans-acting factors directing their transport and translational regulation [69]. Biochemical purification of such complexes via their association with kinesin revealed several nuclear-acquired mRNP proteins, including the pre-mRNA splicing factors TLS and PSF, the mRNA export factor Aly, and hnRNPs A0, A1, A2, D and U. Of these, PSF and hnRNP U were directly confirmed to be required for dendritic localization of a CaMKII␣ reporter [70]. Another protein essential for dendritic targeting of a CaMKII reporter and found to co-purify with neuronal granules is Staufen1 [70,71]. This factor, and the mammalian brain-specific isoform Staufen 2, are well studied double-strand RNA binding proteins required for microtubule-dependent mRNA transport
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in neuronal dendrites [72,73]. The Xenopus Staufen homologue, xStau1, participates in the vegetal localization of Vg1 mRNA [74] and Drosophila Staufen is an essential factor in the localization of oskar, bicoid and prospero mRNAs (reviewed in [22]). In all of these organisms, Staufen proteins display a predominantly, if not exclusively, cytoplasmic distribution, and in Xenopus oocytes, immunoprecipitation experiments suggest that initial xStau1 association with Vg1 mRNA occurs in the cytoplasm [40]. Nonetheless, both mammalian Staufen isoforms have recently been observed in the nucleus suggesting that, at least in mammalian cells, recruitment of this essential transport factor might occur prior to cytoplasmic export [75–77]. 4. Concluding remarks Although we have only begun the scratch the surface of the factors and mechanisms involved in cytoplasmic mRNA transport and localized expression, it is already becoming clear that initiation of this process often involves the loading of factors in the nucleus. In some cases, nuclear pre-assembly of localized mRNPs also provides the elements necessary for silencing translation during the cytoplasmic journey. A clear advantage of nuclear acquisition of translational inhibitors is the elimination of any potential for premature displacement of mRNP transport and/or regulatory factors by a pioneer round of translation immediately upon nucleocytoplasmic export. In other words, it circumvents the need for translational inhibitors to out compete translation initiation factors as the mRNP emerges from the nuclear pore complex. By eliminating the possibility of premature translation, nuclear preassembly of translationally-silenced and transport-competent mRNPs would also minimize inappropriate expression of proteins whose function requires strict localization at the cellular periphery. In view of such a model of nuclear pre-determination, several questions arise. For example, for those factors acquired co-transcriptionally, to what extent is this acquisition dependent on the transcription and RNA processing machineries? Are other factors acquired in specific sub-domains of the nucleus (e.g, the nucleolus or at the nuclear periphery)? To what degree is transport-competency of the mRNP ultimately completed by acquisition of additional factors in the cytoplasm? When additional cytoplasmic factors are required, where does this final cytoplasmic assembly stage occur? Technically, many of these questions may be beyond our current capabilities. Answers to some will undoubtedly require innovative methodological advances in both in situ and biochemical analysis of these highly dynamic complexes. In the coming years it will be of great interest to determine how far reaching is the influence of nuclear nurturing on cytoplasmic mRNP destiny. Acknowledgement Our research is supported by the Howard Hughes Medical Institute.
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Review
The nuclear nurture and cytoplasmic nature of localized mRNPs Corinna Giorgi, Melissa J. Moore ∗ Department of Biochemistry, Howard Hughes Medical Institute, Brandeis University, Waltham, MA 02454, USA Available online 23 January 2007
Abstract From yeast to mammals, evidence has emerged in recent years highlighting the essential role played by the nuclear “history” of a messenger RNA in determining its cytoplasmic fate. mRNA localization, translation and stability in the cytoplasm are often pre-destined in the nucleus, and directed by the composition and architecture of nuclear assembled mRNA–protein complexes. In this review we focus on nuclear-acquired RNA-binding proteins and complexes that participate in determining the journey of localized mRNAs. © 2007 Published by Elsevier Ltd. Keywords: mRNA localization; Exon junction complex; Nuclear mRNA processing
Contents 1. 2.
3.
4.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The exon junction complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. The EJC, translation and NMD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. The EJC and mRNA localization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Other nuclear-acquired mRNA binding proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. ZBP1 proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.1. Ash1 mRNA and associated factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. HnRNP and SR proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.1. HnRNP A2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.2. Squid and Hrp48 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Neuronal transport granules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction A perennial debate among psychologists concerns the varying degrees to which Nature and Nurture contribute to human behavior and destiny. Nature is generally defined as an individual’s innate qualities (i.e., genetic makeup), while Nurture refers to personal experiences (i.e., one’s emotional and physical environment, particularly during prenatal development and childhood). Although historically this debate has swung widely ∗
Corresponding author. Tel.: +1 781 736 2359; fax: +1 781 736 2337. E-mail address: [email protected] (M.J. Moore).
1084-9521/$ – see front matter © 2007 Published by Elsevier Ltd. doi:10.1016/j.semcdb.2007.01.002
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between the two extremes, a more modern view divides human qualities into three broad categories: exclusively genetic (e.g., blood type and eye color), exclusively environmental (e.g., languages spoken and cultural beliefs) and interactional (e.g., height, weight and almost all psychological traits such as IQ). Recent advances in our understanding of gene expression now make it clear that parallel forces are at work at the molecular level determining the behavior and destiny of eukaryotic mRNAs. Here Nature can be defined as the primary sequence of the nascent transcript and Nurture as the nuclear environment in which it is processed and assembled with trans-acting factors into a mature and exportable mRNP. In contrast to the three broad categories of human qualities, however, it now seems that
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virtually all traits and behaviors of eukaryotic mRNAs are interactional. For example, even the most basic property of what protein is encoded by an mRNA is not entirely attributable to the primary sequence of the original transcript. Rather it depends on its splicing and polyadenylation patterns, which in turn are functions of both pre-mRNA sequence and the unique set of trans-acting factors encountered during assembly of the processing machinery. This set of trans-acting factors depends on cell type and cellular environment (e.g., signal transduction pathways) as well as pre-mRNA transcription rate and the intranuclear location of the gene. In this review, we will focus on how a history of nurturing in the nucleus affects subsequent mRNA behavior and destiny in the cytoplasm. Behaviors exhibited by individual mRNAs in the cytoplasm include targeted localization to one or more specific subcellular compartments, variations in where and when the translation apparatus is engaged and differential half-lives. As is the nuclear process of pre-mRNA splicing, all of these cytoplasmic behaviors are interactional; that is, they are a function of both the primary mRNA sequence and the trans-acting factor environment. Emergent themes are (1) that much of what happens to an mRNA in the cytoplasm, including its localization, is pre-destined by trans-acting factors acquired in the nucleus and (2) the intimate association between mRNA localization and translational repression. 2. The exon junction complex Perhaps the best known example of how the nuclear history of an mRNA can affect its cytoplasmic destiny is provided by the exon junction complex (EJC). The EJC is a set of proteins
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deposited on mRNAs as a consequence of pre-mRNA splicing. This deposition occurs ∼20 nts upstream of the site of intron excision and has no apparent mRNA sequence requirement [1]. Although not yet formally proven, it is generally assumed that an EJC is deposited upstream of every exon junction on all spliced mRNAs. Once bound, EJCs travel with the mRNA to the cytoplasm where most are ultimately removed as a consequence of the first or “pioneer” round of translation [2,3]. Prior to their displacement, however, they can act as effectors of almost every aspect of mRNA metabolism including subcellular mRNA localization, mRNA translational efficiency and ultimately mRNA decay (Fig. 1) [4–6]. Structurally, the EJC consists of a stably bound, heterotetrameric core that serves as a binding platform for other more transiently associating factors [7]. At the heart of this core is eIF4AIII, the main RNA anchoring protein [8]. A member of the DEAD-box family of ATP-dependent RNA remodeling proteins, eIF4AIII exhibits ATP-dependent RNA binding activity. During EJC assembly, eIF4AIII presumably becomes locked on the RNA when the Y14:MAGOH heterodimer joins and inhibits its ability to hydrolyze ATP. A fourth core component, MLN51, serves to increase eIF4AIII’s inherent affinity for RNA [9]. Two recent crystal structures of the eIF4AIII:Y14:MAGOH:MLN51 heterotetramer bound to RNA are notable in that they represent the first structures of any DEAD-box protein in complex with multiple binding partners which modulate its activity [10,11]. The list of more peripherally associating EJC proteins includes numerous factors involved in pre-mRNA splicing, mRNA export, nonsense-mediated mRNA decay (NMD) and programmed cell death. However, only a subset of these factors has been shown to remain associated with spliced mRNAs in
Fig. 1. Roles played by the EJC in cytoplasmic metabolism of mRNAs. The EJC has been implicated in aiding mRNA localization, modulating translational efficiency and allowing detection of target mRNAs for the nonsense-mediated decay pathway. P-bodies are cytoplasmic sites of mRNA turnover and mRNA storage.
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the cytoplasm. This subset consists of the splicing factor RNPS1 and the NMD factors Upf2 and Upf3b [7]. RNPS1 was originally characterized as a splicing co-activator capable of increasing the efficiency of pre-mRNA splicing in vitro [12]. It is also a component of the trimeric ASAP complex implicated in the induction of apoptosis [13]. Upf2 and Upf3b, along with Upf1, are central components of the nonsense-mediated mRNA decay pathway (see below). Whereas RNPS1 and Upf3b originally associate with the EJC core in the nucleus, Upf2, a perinuclear protein, presumably joins the complex soon after the spliced mRNA enters the cytoplasm [6].
and Upf2 and Upf3b bound to a downstream EJC [17]. This triggering of NMD can be replicated independent of splicing: Upf and certain EJC factors, if artificially tethered to the 3 UTR of a reporter mRNA, can induce decay of the transcript [19]. In contrast, tethering of the same proteins inside the open reading frame (ORF) increases translational yield, replicating the effects of ORF-bound EJCs in polysome recruitment as above. At present, it is unknown how these disparate positional effects, one enhancing translation and the other leading to mRNA degradation, are mechanistically related. Nonetheless, given that they involve an identical set of proteins, it is tempting to speculate that a connection does exist [6,16,20].
2.1. The EJC, translation and NMD 2.2. The EJC and mRNA localization Initial observations linking pre-mRNA splicing to cytoplasmic mRNA metabolism came from studies comparing the translational efficiency or decay of spliced versus cDNA-derived transcripts. In Xenopus oocytes, more protein is produced in the cytoplasm upon nuclear injection of an intron-containing pre-mRNA than upon injection of the corresponding cDNA transcript ([14] and references therein). This effect is readily observable in mammalian cells as well, where intron-containing and cDNA versions of otherwise identical genes yield significantly different amounts of protein per molecule of cytoplasmic mRNA [5,15]. This influence of splicing on translational yield has been directly linked to EJC deposition, and sedimentation analysis indicated that a greater proportion of spliced mRNAs are polysome-associated than unspliced mRNAs [16]. Though the exact mechanism of how the enhanced polysome association is achieved has yet to be elucidated, one model is that EJCs aid in recruiting translation initiation factors to promote the pioneer round of translation. Another possibility is that, prior to their removal by the pioneer round, EJCs assist in delivering newly exported mRNAs to regions of the cytoplasm (e.g., the cytoskeleton) enriched in ribosomes. Regardless of the mechanism, one potential advantage of such enhanced translation of EJC-carrying mRNAs is that it allows for preferential translation of newly transcribed messages, thus decreasing any delay time between transcriptional activation and protein expression. Whereas EJCs in the coding region can enhance gene expression by increasing translational yield, EJC deposition in the 3 -UTR has the opposite effect of triggering mRNA decay via the NMD pathway. NMD is generally viewed as a protective mechanism allowing cells to eliminate aberrant mRNAs containing pre-mature termination codons (e.g., those produced from mutant alleles or by improper pre-mRNA processing), which might otherwise direct the synthesis of truncated and potentially dominant-negative proteins [17]. Another, and likely as important, function of the NMD pathway is to regulate expression of natural NMD targets. Such natural targets include mRNAs containing upstream open reading frames (uORFs) and those produced from transcripts harboring introns in the 3 -UTR [17,18]. In mammalian cells, a major trigger for the NMD process is a stop codon located >50 nts upstream of at least one exon–exon junction. A proposed model for this type of NMD entails interaction between Upf1 bound to a ribosome stalled at the stop codon
A third known function for the EJC is in localization of oskar mRNA in the developing Drosophila oocyte. Restricted translation of oskar at the posterior pole is essential for germ line and abdomen formation in the future embryo [21]. Oskar mRNA is initially synthesized in the adjacent nurse cell nuclei and then transported into the oocyte through ring canals connecting the nurse cell and oocyte cytoplasms. Transport within the oocyte occurs along microtubules in a plus-ended direction mediated by the motor protein kinesin [22]. Extensive genetic analyses have identified a plethora of trans-acting factors required for translational repression, transport, anchoring and subsequent translational activation of oskar mRNA at the posterior pole. Among the factors required for transport are all four of the EJC core proteins: eIF4AIII, Barentsz, Mago Nashi and Tsunagi (the Drosophila orthologs of MLN51, Magoh and Y14, respectively) [4,23]. All four of these proteins initially accumulate with oskar mRNA at the posterior pole. Although the EJC core factors are essential for proper oskar mRNA localization, it was not initially thought that pre-mRNA splicing had any involvement. This was because chimera experiments had shown that the 3 -UTR of oskar mRNA, which contains no introns, is sufficient to direct posterior pole localization of an intronless reporter when expressed in an otherwise wild type egg chamber [24]. However, this is not the case in egg chambers lacking endogenous oskar mRNA [25]. Under these conditions, proper localization of oskar mRNA expressed from an exogenous transgene requires that the transgene contain at least one intron. This result clearly demonstrates that splicing is required for oskar mRNA localization. Subsequent analysis revealed that the oskar 3 -UTR is capable of dimerization via binding of the trans-acting factor Bruno to its Bruno response elements (BREs) [26]. This nicely accounts for the previous finding that an intronless oskar chimera can be properly localized in the presence of endogenous oskar mRNA, as the chimera could heterodimerize and thereby be transported piggyback style with the endogenous EJC-carrying transcripts. The requirement of EJC deposition for proper oskar expression highlights an emerging connection between mRNA localization and translational repression. Because all three introns in the pre-mRNA lie within the coding region, all oskar EJCs are subject to removal by the pioneer round of translation. Thus, in order to maintain the EJC association necessary
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for its localization, oskar mRNA must not undergo translation prior to arrival at its target destination. While oskar is the clearest example to date of an mRNA requiring translational silencing for localization, more and more examples are emerging of links between these two processes (see below). Thus, oskar may exemplify the generality that localized mRNAs are translationally repressed prior to and during transport. Whereas no direct evidence yet exists for involvement of the EJC in localizing other mRNAs in other organisms or cell types, circumstantial evidence may be pointing in that direction. For example, Magoh, Y14 and MLN51 all localize to the dendrites of mammalian hippocampal neurons [27–29]. These highly polarized cells rely on local translation of dendritically targeted mRNAs to modulate the activity of individual synapses. Within dendrites, MLN51 associates with the known mRNA transport factor Staufen 1 and the abundant transported small RNA, BC1, which has been implicated in the translational regulation of localized mRNAs. Furthermore, data from our own laboratory indicate that eIF4AIII exists in dendritic mRNA localization particles and might be involved in regulating local protein expression at synapses (C.G. and M.J.M., unpublished data). Thus it seems that we have only begun to scratch the surface of the EJC’s involvement in cytoplasmic mRNA metabolism. 3. Other nuclear-acquired mRNA binding proteins Although the EJC has garnered much attention in recent years, it is but one example of an ever-expanding list of nuclearacquired factors that impact cytoplasmic mRNA metabolism [30]. The remainder of this article focuses specifically on those factors whose function can be linked to proper expression of localized mRNAs. 3.1. ZBP1 proteins One of the best examples of nuclear assembly of a transport-competent mRNP involves association of the zipcodebinding-proteins 1 and 2 (ZBP1/2) with -actin mRNA. Members of the ZBP family contain RRM and KH RNAbinding domains and mediate cytoplasmic mRNA localization in a variety organisms and cell types. In chick embryo fibroblasts, neuronal growth cones and rat hippocampal neurons, proper cell structure and motility require asymmetric localization of -actin mRNA driven by ZBP1 (reviewed in [31]). ZBP1 recognizes a 54 nucleotide localization element, the zipcode, in the -actin 3 -UTR. In fibroblasts, mutations in this zipcode or treatment with zipcode-antisense oligonucleotides lead to delocalization of -actin mRNA and alteration of cell motility [32,33]. In mammalian neurons, ZBP1 is required for the dendritic targeting of -actin mRNA, and altered ZBP1 levels affect the density of dendritic filopodia, actin-rich dendritic protusions involved in synaptogenesis [34,35]. ZBP1 is predominantly cytoplasmic. Nonetheless it contains Rev-like nuclear export and nuclear localization sequences, and shuttles between the nucleus and cytoplasm utilizing the Crm1 export pathway [32]. Using a combination of in situ hybridization and immunofluorescence in chicken fibroblasts, Oleynikov
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and Singer demonstrated that ZBP1 associates with -actin mRNA at the site of transcription. Furthermore, impeding ZBP1 nuclear export with Leptomycin B affected cytoplasmic -actin mRNA transport [32]. A second nucleo-cytoplasmic shuttling protein, ZBP2, also binds the -actin zipcode element. Although ZBP2 is predominantly nuclear, a small portion does co-localize with cytoplasmic -actin mRNA in both chick fibroblasts and neurons, suggesting that ZBP2 travels out of the nucleus aboard the mRNA. A direct role of nuclear-acquired ZBP2 in mediating cytoplasmic targeting of -actin mRNA was suggested by the finding that overexpression of a truncated ZBP2 suppresses -actin mRNA localization in fibroblasts and neurons [36]. ZBP2-related proteins include the splicing factor FBP2/KSRP in humans, MARTA1 in rodents, and VgRBP71 in Xenopus. Like ZBP2, both MARTA1 and VgRBP71 are predominantly nuclear. Nonetheless, they have been implicated in cytoplasmic mRNA localization of Map2 mRNA in mammalian dendrites and of several localized mRNAs in Xenopus oocytes, respectively. This suggests a conserved role for nuclearacquired ZBP2-like proteins in cytoplasmic mRNA localization [37,38]. A link between nuclear ZBP acquisition and cytoplasmic mRNA localization was also recently reported in Xenopus oogenesis. Vg1 and VegT are two maternal mRNAs that localize at the vegetal pole and have been implicated in mesoderm and endoderm specification of the embryo. Their vegetal pole localization is directed in part by the trans-acting factors Vg1RBP60/hnRNPI and VERA/Vg1RBP, the Xenopus homologue of ZBP1. These factors bind to 3 -UTR localization elements (LEs) in the transported mRNAs and mutations in the LEs that disrupt protein binding lead to mislocalization of the transcripts [39]. Immunoprecipitation of tagged Vg1RBP60/hnRNPI and VERA/Vg1RBP revealed that both proteins associate with Vg1 and VegT mRNAs in the nucleus as well as the cytoplasm [40]. A recent report by the Singer lab [41] demonstrates that in addition to directing -actin mRNA to the leading edge of fibroblasts, ZBP1 is also involved in repressing its translation both in vitro and in neuroblastoma cells. ZBP1 therefore seems to couple transport with translational repression of -actin mRNA. A combination of in vitro and in vivo approaches also provided evidence for how the translational repression can be reversed once the mRNA is localized. The suggested mechanism involves phosphorylation of ZBP1 by the tyrosine kinase Src resulting in diminished affinity for the bound mRNA and its consequent emancipation from translational repression. SRC involvement in regulating asymmetric -actin mRNA expression was also reported in a recent study linking localization of both species to Ca2+ -dependent growth cone guidance in Xenopus laevis spinal neurons [42]. 3.1.1. Ash1 mRNA and associated factors One of the best-understood examples of active mRNA transport (i.e., requiring molecular motors) is provided by S. cerevisiae ASH1 mRNA [43]. During cell division, ASH1 mRNA migrates from the mother cell to the bud tip where its localized
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expression is key for suppressing mating type switching in the daughter cell. ASH1 mRNA localization requires direct association of the RNA-binding protein She2, a nucleocytoplasmic shuttling protein, with specific mRNA sequence elements. She2 then recruits She3, which in turn interacts with She1, a type V myosin. In addition to its key role in localization, She2 binding to three sites on ASH1 mRNA coding region is required for translational repression of the mRNA during transport. Mutant mRNAs in which She2 binding sites are moved to the 3 UTR retain the ability to localize but are translationally derepressed during transport [44]. The nuclear connection in this system comes in the form of Loc1, a nuclear restricted protein required for the initial recruitment of She2 to ASH1 mRNA [45]. 3.2. HnRNP and SR proteins Heterogeneous nuclear ribonucleoproteins (hnRNPs) comprise a diverse set of proteins originally identified by their association with hnRNA. Characteristic of this class is the presence of one or more RNA binding domains, including RRMs, RGG and KH domains [46]. Another major class of RNAbinding factors is the SR proteins, which, in addition to a N-terminal RRM, typically have a C-terminal domain rich in RS (arginine-serine) repeats. SR proteins are subject to dynamic methylation and/or phosphorylation within the RGG and RS regions. Such modifications, which can be compartment limited, can alter the RNA binding properties of the modified protein [47–52]. HnRNPs and SR proteins can also be subclassified according to whether or not they shuttle in and out of the nucleus [53,54]. It is the shuttling class that is relevant for the discussion below. Generally these factors associate with mRNA in the nucleus, many co-transcriptionally, and, like the EJC, accompany mRNAs to the cytoplasm where they modulate mRNA localization, translational efficiency and mRNA stability. Many also have important and distinct functions in the nucleus in addition to their cytoplasmic roles [46,47,55]. 3.2.1. HnRNP A2 One of the best understood hnRNPs involved in mRNA localization is hnRNP A2 (a.k.a. hnRNP A/B). HnRNP A2 is required for the localization of myelin basic protein mRNA (MBP mRNA) to myelin-forming processes in mammalian oligodendrocytes [56]. Necessary and sufficient is the 11 nucleotide A2 response element (A2RE) located in the MBP mRNA 3 UTR. Introduction of this element into a non-localized GFP reporter is capable of driving its extrasomatic localization when microinjected in oligodendrocytes [57]. Further, when the A2RE contains mutations interfering with hnRNP A2 binding, the mutant mRNA fails to localize properly. Finally, microinjection of either antibodies or antisense oligonucleotides targeting hnRNP A2 adversely affects trafficking of microinjected A2RE RNAs ([58] and references therein). A2RE-like sequences are also found in several other mRNAs, including three HIV transcripts and the dendritically localized CaMKII␣, Arc, Neurogranin and MAP2 mRNAs, although only a subset of these elements has been tested in an RNA localization assay [57–60].
Beyond its role in mRNA localization, recent data also suggest a possible involvement of hnRNP A2 in modulating translation of the cargo mRNA. Within A2RE-mRNP transport granules in oligodendrocytes, hnRNP A2 interacts and co-localizes with hnRNP E, a predominantly cytoplasmic translational repressor. Functional assays revealed that hnRNP A2 serves to recruit hnRNP E to A2RE-containing reporter mRNAs in vivo, resulting in translational inhibition [61]. 3.2.2. Squid and Hrp48 In Drosophila, the hnRNPs Squid and Hrp48 (closely related to mammalian hnRNP A1 and A2, respectively) are involved in cytoplasmic mRNA localization during oogenesis (reviewed in [21]). Squid binds directly to the 3 -UTR of gurken mRNA and this association is required for proper localization of gurken to the dorso-anterior corner of the oocyte [62]. Hrp48 binding to oskar mRNA in both the 5 and 3 portions of the transcript is essential for the proper delivery of oskar to the posterior pole [63]. In an interesting twist, recent work indicates that localization of oskar mRNA to the posterior pole also requires Squid, and, conversely, Hrp48 participates in localization of gurken mRNA to the dorso-anterior corner [64,65]. This suggests that both of these proteins act as transport competency factors, but neither determines the ultimate destination of the mRNA within the oocyte. Drosophila Hrp48 and Squid also participate in repressing translation of oskar and gurken mRNAs, respectively [66], offering another example of the tight coupling between cytoplasmic targeting and translational repression of localized mRNA. While Squid seems to bind gurken mRNA in the nucleus [62], it is at present unclear where Hrp48 first associates with oskar mRNA. In contrast to its mammalian homolog (hnRNP A2), Hrp48 is predominantly cytoplasmic [67]. Nonetheless, a small fraction does exists in the nucleus where it has been shown to participate in alternative splicing of nascent mRNAs ([68] and references therein), so Hrp48 could certainly be a nuclearacquired mRNP factor. 3.3. Neuronal transport granules HnRNPs have also been found in neuronal transport granules. These large macromolecular assemblies are found in dendrites and include dendritically targeted mRNAs plus numerous trans-acting factors directing their transport and translational regulation [69]. Biochemical purification of such complexes via their association with kinesin revealed several nuclear-acquired mRNP proteins, including the pre-mRNA splicing factors TLS and PSF, the mRNA export factor Aly, and hnRNPs A0, A1, A2, D and U. Of these, PSF and hnRNP U were directly confirmed to be required for dendritic localization of a CaMKII␣ reporter [70]. Another protein essential for dendritic targeting of a CaMKII reporter and found to co-purify with neuronal granules is Staufen1 [70,71]. This factor, and the mammalian brain-specific isoform Staufen 2, are well studied double-strand RNA binding proteins required for microtubule-dependent mRNA transport
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in neuronal dendrites [72,73]. The Xenopus Staufen homologue, xStau1, participates in the vegetal localization of Vg1 mRNA [74] and Drosophila Staufen is an essential factor in the localization of oskar, bicoid and prospero mRNAs (reviewed in [22]). In all of these organisms, Staufen proteins display a predominantly, if not exclusively, cytoplasmic distribution, and in Xenopus oocytes, immunoprecipitation experiments suggest that initial xStau1 association with Vg1 mRNA occurs in the cytoplasm [40]. Nonetheless, both mammalian Staufen isoforms have recently been observed in the nucleus suggesting that, at least in mammalian cells, recruitment of this essential transport factor might occur prior to cytoplasmic export [75–77]. 4. Concluding remarks Although we have only begun the scratch the surface of the factors and mechanisms involved in cytoplasmic mRNA transport and localized expression, it is already becoming clear that initiation of this process often involves the loading of factors in the nucleus. In some cases, nuclear pre-assembly of localized mRNPs also provides the elements necessary for silencing translation during the cytoplasmic journey. A clear advantage of nuclear acquisition of translational inhibitors is the elimination of any potential for premature displacement of mRNP transport and/or regulatory factors by a pioneer round of translation immediately upon nucleocytoplasmic export. In other words, it circumvents the need for translational inhibitors to out compete translation initiation factors as the mRNP emerges from the nuclear pore complex. By eliminating the possibility of premature translation, nuclear preassembly of translationally-silenced and transport-competent mRNPs would also minimize inappropriate expression of proteins whose function requires strict localization at the cellular periphery. In view of such a model of nuclear pre-determination, several questions arise. For example, for those factors acquired co-transcriptionally, to what extent is this acquisition dependent on the transcription and RNA processing machineries? Are other factors acquired in specific sub-domains of the nucleus (e.g, the nucleolus or at the nuclear periphery)? To what degree is transport-competency of the mRNP ultimately completed by acquisition of additional factors in the cytoplasm? When additional cytoplasmic factors are required, where does this final cytoplasmic assembly stage occur? Technically, many of these questions may be beyond our current capabilities. Answers to some will undoubtedly require innovative methodological advances in both in situ and biochemical analysis of these highly dynamic complexes. In the coming years it will be of great interest to determine how far reaching is the influence of nuclear nurturing on cytoplasmic mRNP destiny. Acknowledgement Our research is supported by the Howard Hughes Medical Institute.
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