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From the Nucleus Toward the Cell Periphery: a Guided Tour for mRNAs Brigitte M. Jockusch,1 Stefan Hüttelmaier,1,2 and Susanne Illenberger1 1 Cell Biology, Zoological Institute, Technical University of Braunschweig, D-38092 Braunschweig, Germany; and 2Albert Einstein College of Medicine, New York, New York 10461 T issue generation during embryogenesis and wound healing involves cell proliferation as well as cellular differentiation. The latter comprises a variety of highly complex processes, culminating in structural and functional polarity of the tissue-forming cells. The most important steps are differential gene activation and transcription, RNA splicing and maturation, and, finally, site-specific translation of tissue-specific proteins. Recent evidence suggests that these steps may be coupled, which would increase the efficiency of the assembly of peripheral protein complexes. However, temporal as well as spatial coupling requires mediators. For example, how are nuclear processes, like RNA generation and processing, wired to steps occurring at the cell periphery, like the assembly of plasma membrane-associated protein complexes by site-specific protein synthesis? The discovery of specific “dual-compartment” proteins that participate in nuclear as well as in peripheral processes may provide an answer to this question. Such proteins could act as tour guides for mRNAs traveling from one compartment to the other. The guiding process itself, however, is complex and far from understood today. Transport vehicles, traffic, and cargo delivery Site-specific localization of mRNA in cells, as a result of either directed transport, concentration by a specific anchoring mechanism, or region-specific protection from degradation, has been observed in a large number of cases, in particular in oocytes and early embryos of vertebrates and insects, but it has also been observed in yeast, where these mechanisms result in targeting of proteins engaged in cell and tissue polarization or the determination of cell fate (1, 15). To better understand the mechanism and the biological significance of the directed transport of mRNAs in all of these systems, one would need to know the exact composition of their cytoplasmic cages (“locasomes”; Ref. 3), the relation between specific cargo mRNAs and their locasomes, the nature of the tracks, and the linker as well as the motor proteins that might drive the polarized transport. Regarding the tracks, there is good evidence that mRNA movement utilizes the cytoskeleton (8). Of the three major cytoskeletal filament systems expressed in animal cells [intermediate filaments, microtubules, and micro0886-1714/03 5.00 © 2003 Int. Union Physiol. Sci./Am. Physiol. Soc. www.nips.org Downloaded from http://physiologyonline.physiology.org/ by 10.220.32.247 on June 15, 2017 RNA processing, directed transport along cytoskeletal tracks, and site-specific translation of mRNA at the cell periphery are considered discrete steps in the generation of microfilament-membrane adhesion complexes. A recently identified member of the heterogeneous nuclear ribonucleoprotein family, raver1, may couple these steps and contribute to the assembly and maintenance of these structures. filaments] two, namely microtubules and microfilaments, participate in the directed transport of mRNAs. The mRNA-containing protein complexes are tied to cytoskeletal fibers, transported along them, and also translated when still tethered to these structures (e.g., Ref. 4). Apparently, the choice of the tracks is not so much dependent on the nature of the mRNA transported but on the cell type, and a switch from one to the other cytoskeletal track system also seems possible (1, 15). Accordingly, both types of cytoskeletal motor enzymes, i.e., kinesins and myosins, may be involved. Within this field, the transport of cytoskeletal mRNAs raises a particularly interesting question: do the cytoskeletal elements serve as tracks to move their own mRNAs and thus contribute to the directed translation of their own components? This might be an efficient mechanism in the assembly of peripheral structures, such as microfilament-plasma membrane attachment sites in adhesive junctions that play a crucial role in tissue generation. Having reached their destination and at the end of their motor-assisted journey, at least some components of the locasomes must be anchored and, finally, their cargo mRNA should be translated. In this context, the identification of the elongation factor EF1 as an actin filament- and -actin mRNA-binding protein is noteworthy, because it may couple anchoring of actin mRNA to the cytoskeletal fibers with translation and thus allow for cotranslational assembly of cytoskeletal structures in distal parts of the cell (12). b-Actin is the actin isoform associated with dynamics and motility of the cell periphery, and -actin mRNA was seen concentrated at the leading edge of locomoting fibroblasts (10) and at the tips of neurites (2). The interaction between this mRNA and proteins engaged in its transport through the cytoplasm has been studied in detail. Within the 3'-untranslated region, a 54-nt motif, the “zip code,” was found to interact with several zip code-binding proteins, and this interaction seems essential for transport, localization, and translation of actin mRNA in fibroblasts and neurons (6). One of these proteins, ZBP2, is an avian ortholog of the human protein KHtype splicing regulatory protein (KSRP), is a member of the heterogeneous nuclear ribonucleoprotein (hnRNP) family (see below), and is predominantly, but not exclusively, located in the nucleus (6). Another interesting case of complexes News Physiol Sci 18: 711, 2003; 10.1152/nips.01413.2002 7 The common view on how PTB/hnRNPI and related proteins can achieve so many functions is of course that they associate with different ligand proteins, contributing to discrete multiprotein complexes that then participate in only a few or even one of these functions. Indeed, hnRNPs form homo- and heterooligomeric complexes with other members of the family, but a complete catalog of their ligands is still missing. It is also not known whether, in general, shuttling hnRNPs are packaged and transported along with the mRNAs, and, if so, whether these are the same hnRNPs that already participated in the generation of the particular mRNA that is cotransported. Raver1, an hnRNP protein with unique properties Downloaded from http://physiologyonline.physiology.org/ by 10.220.32.247 on June 15, 2017 The recent discovery of raver1 (7), another hnRNPI protein, FIGURE 1. Raver1 colocalizes with pyrimidine tract-binding protein (PTB)/heterogeneous nuclear ribonucleoprotein I (hnRNPI) in perinucleolar bodies of myoblasts. Double fluorescence images obtained with antibodies against PTB/hnRNPI (left) and raver1 (middle) show that, in undifferentiated C2C12 myoblasts, both proteins reside within the nuclear matrix but not within nucleoli (black spots). Both are concentrated in the perinucleolar bodies (arrowheads), and their exact colocalization is demonstrated in the overlay images (right). Bars = 10 m. (Reprinted with permission from Ref. 7). between mRNAs and peripheral proteins is represented by recent data demonstrating that PABP1, a protein that binds to and stabilizes the poly(A) tail of a variety of mRNAs, forms high-affinity complexes with paxillin, a microfilament adaptor protein involved in the regulation of cell motility (20). The hnRNP family: multitalented tour guides These findings indicate that the multiple steps from nuclear RNA maturation to peripheral translation may be guided by proteins that are multifunctional and act as chaperones for mRNAs on their journey from the nucleus to the periphery. In the search for candidates mediating between mRNAs and protein components of transport particles, a number of RNAbinding proteins of the hnRNP family (5) have gained much attention. The hnRNPs display protein-specific subsets of conserved RNA-binding domains, including RNA-recognition motifs (RRMs), heterogeneous nuclear ribonucleotide protein K (hnRNPK) homology domains (KH domains), and glycinearginine-rich domains that are involved in mediating specific protein-RNA and protein-protein interactions. These proteins bind to premature and mature mRNA and are associated with several steps of mRNA processing, sorting, and splice control. Although most hnRNPs are nuclear resident proteins, some can shuttle between the nucleus and the cytoplasm (16), and they seem to execute different tasks in these compartments. For the pyrimidine tract-binding protein (PTB/hnRNPI), which is one of the most intensely studied proteins of the hnRNP family, the proposed functions in the nucleus involve controlling alternative pre-mRNA splicing and thus tissue-specific translation of a variety of different proteins (18). In addition, the same protein seems to participate in nuclear export of mature mRNAs, their directed transport within the cytoplasm, and even translation and regulation of mRNA stability (11). 8 News Physiol Sci • Vol. 18 • February 2003 • www.nips.org FIGURE 2. During myogenesis, raver1 moves from a nuclear position to the cell periphery. Double-fluorescence images obtained with antibodies against raver1 and the transcription factor myocyte enhancer factor (MEF)-2 reveal that, in undifferentiated mouse C2C12 myoblasts (MB), both proteins reside exclusively in the nucleus (A). After induction of differentiation and myoblast fusion, raver1 leaves this position and is also seen in the sarcoplasma, whereas MEF-2 stays in the nuclear compartment (B). When differentiation has proceeded to cross-striated myotubes (MT), raver1 is seen at cytoplasmic structures that probably correspond to costameres in the fully differentiated muscle (cf. Fig. 3), whereas the nuclei are devoid of raver1 signals (C; at right is shown a higher magnification from the images at left). Bars = 10 m (Reprinted with permission from Ref. 7). Downloaded from http://physiologyonline.physiology.org/ by 10.220.32.247 on June 15, 2017 has shed new light on the function of members of this protein family, showing that their catalog of functions can still be larger. The sequence of the 80-kDa polypeptide of raver1 displays three NH2-terminally located RRMs with homology to other members of the hnRNP family, in particular PTB/hnRNPI, two bona fide nuclear location sequences (NLS), and one REV protein-like nuclear export sequence (NES). These motifs are indeed considered a telltale for an hnRNP protein, and it was shown in vitro that raver1 can also form heterocomplexes with other hnRNPs, for example with PTB/hnRNPI (7). In many different cell types, raver1 was found located in the nucleus, with a high local concentration in morphologically discrete nuclear particles. In heterokaryon assays with hybrids of mouse and raver1-transfected human cells, raver1 was seen to shuttle between the nucleus and the cytoplasm. This ability is probably based on the presence of both active NLS and NES sequences. Shuttling of raver1 is not confined to transfected cells but also occurs in nonmanipulated cells, where it is apparently correlated with tissue differentiation: in the nucleus of undifferentiated myoblasts, raver1 colocalizes with PTB/hnRNPI in the perinucleolar bodies (Fig. 1). These are small, distinct structures adjacent to nucleoli that are enriched in hnRNPs and believed to participate in the generation of mRNAs (17). It is probably in this cellular region that raver1 interacts with PTB/hnRNPI and exerts its hnRNP-like activity; preliminary data from an in vitro assay indicate that raver1 contributes as a coregulator to alternative splicing of actinin pre-mRNA as induced by PTB/hnRNPI (N. Gromak, S. Hüttelmaier, and C. Smith, personal communication). During myogenesis, raver1 starts migration; it changes its location from a purely nuclear position in myoblasts to the presumptive costameres of differentiating myotubes (Fig. 2). Finally, in mature muscle, raver1 is associated with microfilament membrane attachment sites, such as costameres of skeletal muscle (Fig. 3; Ref. 7) and the intercalated disks of cardiomyocytes (S. Illenberger and B. M. Jockusch, unpublished observations). How is raver1 targeted to these sites? In a yeast two-hybrid analysis, raver1 was identified as a direct ligand for three structural proteins of the intercalated disk: metavinculin, vinculin, and -actinin. Whereas metavinculin, a larger-splice form of vinculin, is only expressed in smooth, skeletal, and cardiac muscle [in the latter apparently contributing to the force transmission in the beating heart (14)], vinculin and actinin are ubiquitous structural components of adhesive junctions. All three proteins may thus recruit raver1 to (muscular) microfilament attachment sites. Biochemical and immunoprecipitation studies demonstrated that raver1 binds directly to these proteins and that ternary complexes comprising raver1, vinculin, and -actinin could be precipitated from cell lysates (7). Comparing the binding properties of the fulllength raver1 with those of appropriate deletion fragments revealed that the NH2-terminal half of raver1 harbors the binding sites for vinculin/metavinculin, whereas the COOH-terminal half of the molecule interacts with -actinin (Fig. 4A). Furthermore, it turned out that raver1 also colocalizes with vinculin and -actinin in microfilament-plasma membrane attachment sites of adhesion complexes in epithelial or fibroblastic cells (7). Hence, this hnRNP protein is a ubiquitous FIGURE 3. Raver1 colocalizes with vinculin/-actinin at microfilament attachment sites in mouse skeletal muscle. The immunofluorescence images show frozen sections of mouse M. soleus after double staining with antibodies against raver1 (A and D), vinculin (B), and -actinin (E). The overlays (C and F) reveal a substantial colocalization of raver1 with both of these proteins, which are structural components of costameres, the microfilament attachment sites connecting the periphery of Z-lines with the sarcolemma. Bars = 10 m (Reprinted with permission from Ref. 7). component of a wide variety of adhesive structures, which suggests that it is an important factor in their assembly. What is the significance of raver1 targeting to microfilament-associated adhesion complexes, especially during myogenesis? Given a possible role of raver1 in mRNA transport and targeting, it is interesting to note that vinculin mRNA was located at costameres in embryonic muscle (13); hence, the differentiation-associated migration of raver1 to costameres and intercalated disks may reflect its role in vinculin mRNA transport, but there are as yet no data on either vinculin RNA splicing or transport. On the basis of these data, one might postulate multiple roles of raver1 in assembling adhesion complexes. Nuclear residence of raver1 would be achieved by functional NLS, and the protein might be retained in this position by tethering to nascent transcripts, as was suggested for PTB/hnRNPI (9) and another shuttling hnRNP, A1 (19). Once transcription and splicing of a particular subset of mRNAs (specific for adhesion complexes) is finished, raver1 might use its NES to leave its position and travel to the cytoplasm, taking the corresponding mRNAs along. Raver1, together with the spliced mRNAs, would then be incorporated into locasomes and transported News Physiol Sci • Vol. 18 • February 2003 • www.nips.org 9 Downloaded from http://physiologyonline.physiology.org/ by 10.220.32.247 on June 15, 2017 FIGURE 4. The putative role of raver1 in mRNA processing and translation of cell adhesion proteins. A: deduced amino acid sequence and ligand-binding motifs of raver1. The polypeptide sequence is shown in orange. Sequence motifs with a recognized function as deduced from databases are indicated as follows: the two nuclear location signals are shown in black, the single nuclear export signal is shown in white, and the three RNA-recognition motifs are hatched. Binding regions for the three protein ligands identified so far are shown by double-headed arrows underneath the sequence diagram. The numbers refer to amino acids. B: how raver1 may act as a tour guide during the formation of adhesion complexes. In the nucleus, raver1 participates in RNA processing within spliceosomes, probably in concert with PTB/hnRNPI. Both proteins may also be part of the export complex, transporting the mature mRNA into the cytoplasm. In specialized locasomes that have yet to be characterized in their molecular composition, raver1 together with other cofactors is transported via cytoskeletal elements to peripheral cell adhesion sites. By binding to -actinin and vinculin, integral proteins of attachment plaques, raver1 may help anchor the mRNA to these structures and allow site-specific translation, thus contributing to the assembly of cell adhesion complexes. 10 News Physiol Sci • Vol. 18 • February 2003 • www.nips.org along cytoskeletal elements. Once these particles reach their destination at the cell periphery, translation is initiated, the protein products are tethered to raver1 in this position, and such complexes contribute to the assembly of adhesion structures in muscle and nonmuscle cells. The model shown in Fig. 4B emphasizes the most important steps in such a process. 5. 6. 7. Conclusions We apologize to those authors whose publications could not be cited due to lack of space. Our own work referred to in this article was supported by the Deutsche Forschungsgemeinschaft. 8. 9. 10. 11. 12. 13. 14. 15. 16. References 1. Bassell GJ, Oleynikov Y, and Singer RH. The travels of mRNAs through all cells large and small. FASEB J 13: 447 454, 1999. 2. Bassell GJ, Zhang H, Byrd AL, Femino AM, Singer RH, Taneja KL, Lifshitz LM, Herman IM, and Kosik KS. Sorting of beta-actin mRNA and protein to neurites and growth cones in culture. J Neurosci 18: 251 265, 1998. 3. Bertrand B, Pascal C, Schaefer M, Shenoy SM, Singer RH, and Long RM. Localization of ASH1 mRNA particles in living yeast. Mol Cell 2: 437 445, 1998. 4. Cervera M, Dreyfuss G, and Penman S. Messenger RNA is translated 17. 18. 19. 20. Downloaded from http://physiologyonline.physiology.org/ by 10.220.32.247 on June 15, 2017 Although many details of this model (Fig. 4B) have yet to be proven, the discovery of raver1 emphasizes that until now the view of the role of hnRNPs in directed mRNA transport and site-directed translation of membrane-associated microfilament proteins was incomplete. In addition to the multiple functions already attributed to various members of this family, a functional task in the assembly of cell contact structures of differentiating cells has to be added. Proteins like raver1 may spatially and temporally coordinate the different steps like RNA splicing, guide the directed mRNA transport, and contribute to the immobilization of the translated product. 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Getting the message across: the intracellular localization of mRNAs in higher eukaryotes. Annu Rev Cell Dev Biol 17: 569 614, 2001. Pinol-Roma S and Dreyfuss G. Shuttling of pre-mRNA binding proteins between nucleus and cytoplasm. Nature 355: 730 732, 1992. Spector DL. Nuclear domains. J Cell Sci 114: 2891 2893, 2001. Valcarcel J and Gebauer F. Post-transcriptional regulation: the dawn of PTB. Curr Biol 7: R705 R708, 1997. Vautier D, Chesne P, Cunha C, Calado A, Renard JP, and Carmo-Fonseca M. Transcription-dependent nucleocytoplasmic distribution of hnRNP A1 protein in early mouse embryos. J Cell Sci 114: 1521 1531, 2001. Woods AJ, Roberts MS, Choudhary J, Barry ST, Mazaki Y, Sabe H, Morley SJ, Critchley DR, and Norman JC. Paxillin associates with poly(A)-binding protein at the dense endoplasmic reticulum and the leading edge of migrating cells. J Biol Chem 277: 6428 6437, 2002. News Physiol Sci • Vol. 18 • February 2003 • www.nips.org 11