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• W2064848035 abstract "Mammalian Staufen2 (Stau2), a brain-specific double-stranded RNA-binding protein, is involved in the localization of mRNA in neurons. To gain insights into the function of Stau2, the subcellular localization of Stau2 isoforms fused to the green fluorescence protein was examined. Fluorescence microscopic analysis showed that Stau2 functions as a nucleocytoplasmic shuttle protein. The nuclear export of the 62-kDa isoform of Stau2 (Stau262) is mediated by the double-stranded RNA-binding domain 3 (RBD3) because a mutation to RBD3 led to nuclear accumulation. On the other hand, the shorter isoform of Stau2, Stau259, is exported from the nucleus by two distinct pathways, one of which is RBD3-mediated and the other of which is CRM1 (exportin 1)-dependent. The nuclear export signal recognized by CRM1 was found to be located in the N-terminal region of Stau259. These results suggest that Stau2 may carry a variety of RNAs out of the nucleus, using the two export pathways. The present study addresses the issue of why plural Stau2 isoforms are expressed in neurons. Mammalian Staufen2 (Stau2), a brain-specific double-stranded RNA-binding protein, is involved in the localization of mRNA in neurons. To gain insights into the function of Stau2, the subcellular localization of Stau2 isoforms fused to the green fluorescence protein was examined. Fluorescence microscopic analysis showed that Stau2 functions as a nucleocytoplasmic shuttle protein. The nuclear export of the 62-kDa isoform of Stau2 (Stau262) is mediated by the double-stranded RNA-binding domain 3 (RBD3) because a mutation to RBD3 led to nuclear accumulation. On the other hand, the shorter isoform of Stau2, Stau259, is exported from the nucleus by two distinct pathways, one of which is RBD3-mediated and the other of which is CRM1 (exportin 1)-dependent. The nuclear export signal recognized by CRM1 was found to be located in the N-terminal region of Stau259. These results suggest that Stau2 may carry a variety of RNAs out of the nucleus, using the two export pathways. The present study addresses the issue of why plural Stau2 isoforms are expressed in neurons. The localization of mRNA to defined subcellular regions enables the spatial and temporal control of gene expression (1Kiebler M.A. DesGroseillers L. Neuron. 2000; 25: 19-28Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 2Jansen R.P. Nat. Rev. Mol. Cell. Biol. 2001; 2: 247-256Crossref PubMed Scopus (289) Google Scholar, 3Saxton W.M. Cell. 2001; 107: 707-710Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 4Kloc M. Zearfoss N.R. Etkin L.D. Cell. 2002; 108: 533-544Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). In neurons, the dendritic transport of certain mRNAs and the subsequent local synthesis of proteins are thought to play a role in neuronal plasticity. Mammalian Staufen2 (Stau2), 1The abbreviations used are: Stau2, Staufen2; dsRNA, double-stranded RNA; GFP, green fluorescence protein; EGFP, enhanced GFP; RBD, double-stranded RNA-binding domain; NES, nuclear export signal; MBP, maltose-binding protein; LMB, leptomycin B; PBS, phosphate-buffered saline.1The abbreviations used are: Stau2, Staufen2; dsRNA, double-stranded RNA; GFP, green fluorescence protein; EGFP, enhanced GFP; RBD, double-stranded RNA-binding domain; NES, nuclear export signal; MBP, maltose-binding protein; LMB, leptomycin B; PBS, phosphate-buffered saline. a dsRNA-binding protein, was shown to be a homolog of Droshophila melanogaster Staufen (dmStau) (5Buchner G. Bassi M.T. Andolfi G. Ballabio A. Franco B. Genomics. 1999; 62: 113-118Crossref PubMed Scopus (27) Google Scholar, 6Tang S.J. Meulemans D. Vazquez L. Colaco N. Schuman E. Neuron. 2001; 32: 463-475Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 7Duchaine T.F. Hemraj I. Furic L. Deitinghoff A. Kiebler M.A. DesGroseillers L. J. Cell Sci. 2002; 115: 3285-3295PubMed Google Scholar). It has been shown that dmStau anchors bicoid mRNA at the anterior of the oocyte and oskar mRNA to the posterior during early development (8St. Johnston D. Beuchle D. Nusslein Volhard C. Cell. 1991; 66: 51-63Abstract Full Text PDF PubMed Scopus (495) Google Scholar, 9Ferrandon D. Elphick L. Nusslein Volhard C. St. Johnston D. Cell. 1994; 79: 1221-1232Abstract Full Text PDF PubMed Scopus (350) Google Scholar). Mammalian Stau2 is mainly expressed in the brain and is involved in mRNA transport in neurons (6Tang S.J. Meulemans D. Vazquez L. Colaco N. Schuman E. Neuron. 2001; 32: 463-475Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 7Duchaine T.F. Hemraj I. Furic L. Deitinghoff A. Kiebler M.A. DesGroseillers L. J. Cell Sci. 2002; 115: 3285-3295PubMed Google Scholar). Stau2 exists in the somatodendritic compartment of cultured hippocampal neurons. In dendrites, Stau2 associates with RNA granules that contain other RNA-binding proteins, ribosomal subunits, translation factors, and motor proteins (10Ohashi S. Koike K. Omori A. Ichinose S. Ohara S. Kobayashi S. Sato T.A. Anzai K. J. Biol. Chem. 2002; 277: 37804-37810Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 11Mallardo M. Deitinghoff A. Muller J. Goetze B. Macchi P. Peters C. Kiebler M.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2100-2105Crossref PubMed Scopus (140) Google Scholar). RNA granules are transported in dendrites via microtubules (12Kohrmann M. Luo M. Kaether C. DesGroseillers L. Dotti C.G. Kiebler M.A. Mol. Biol. Cell. 1999; 10: 2945-2953Crossref PubMed Scopus (252) Google Scholar). Therefore, RNA granules are thought to play an important role in the targeting of RNA in neurons, functioning as cellular trafficking machinery (13Knowles R.B. Sabry J.H. Martone M.E. Deerinck T.J. Ellisman M.H. Bassell G.J. Kosik K.S. J. Neurosci. 1996; 16: 7812-7820Crossref PubMed Google Scholar), although the formation process by which this macromolecular complex is formed is currently unknown. In addition, it is known that Stau2 has at least three splicing variants with molecular masses of 62, 59, and 52 kDa (7Duchaine T.F. Hemraj I. Furic L. Deitinghoff A. Kiebler M.A. DesGroseillers L. J. Cell Sci. 2002; 115: 3285-3295PubMed Google Scholar). However, the issue of whether these isoforms are functionally different has not been resolved. Because of this, the questions arise as to why such Stau2 isoforms are present in cells and whether each isoform functions divergently. To answer these questions, we investigated the intracellular dynamic behavior of two isoforms of Stau2. In this report, we show that Stau2 functions as a nucleocytoplasmic shuttle protein. In addition, we demonstrate that these two isoforms are distinctly exported from the nucleus, suggesting that each isoform may carry different sets of RNAs out of the nucleus. Cloning of Rat Staufen259—Using information on the expressed sequence tags, the rat Staufen259 was cloned from a rat brain cDNA library (Clontech) by PCR using the following primers: forward, 5′-CCGGACCATGCTTCAGATAAATCAGATGTTTTCGGTGC-3′, and reverse, 5′-ATGGATCCTAGATGACCGACTTTGATTTCTTGCAGTCCTG-3′. The PCR product was digested with BamHI, cloned into the pEGFP-C1 vector (Clontech), and sequenced. Constructions—Full-length rat Stau262 was cloned from a rat brain cDNA library by PCR using the following primers: forward, 5′-CCGGATCCATGGCAAATCCCAAAGAGAAAACTCCAATGTGTCTG-3′, and reverse, 5′-ATGGATCCTAGATGACCGACTTTGATTTCTTGCAGTCCTG-3′. The PCR product was digested with BamHI and cloned into the pEGFP-C1 vector. Using the following primers: forward, 5′-CCGAATTCTCTGAAATAAGCTTAGTGTTTGAAATTG-3′ and reverse, 5′-TTGGATCCAAGCTCCTGTAAGACCG-3′; forward, 5′-GTGAATTCAAGAAACTCCCACCTCTTCCTG-3′ and reverse, 5′-TAGGATCCGAGCTGTAACAGCATAGCTTCTG-3′; forward, 5′-TAGAATTCCCCATTAGCCGCCTGGCTC-3′ and reverse, 5′-TAGGATCCGAGCTGTAACAGCATAGCTTCTGA-3′, the deletion mutants Stau262-(208–272), Stau262-(273–373), and Stau262-(308–373) were amplified from Stau262-pEGFP-C1 by PCR, respectively. The PCR products were digested with EcoRI and BamHI and cloned into the pEGFP-C2 vector. The deletion mutant Stau262-(1–242) was constructed from Stau262-pEGFP-C1 by cutting out the fragments of XmaI-XmaI and ligation into the pEGFP-C1 vector. Stau262-(1–137) was constructed from Stau262-pEGFP-C1 by digestion with HpaI and EcoRV, and the larger fragment was blunted and self-ligated. Stau262-(138–242) was constructed from Stau262-(1–242)-pEGFP-C1 by cutting out the fragments of EcoRI-BamHI and ligated into the pEGFP-C2 vector. Stau262-(243–571) was constructed from Stau262-pEGFP-C1 by digestion with SmaI and KpnI, and the larger fragment was blunted and self-ligated. Stau262-(243–317) was constructed from Stau262-(243–571)-pEGFP-C1 by cutting out the fragments of EcoRI-HaeIII and ligated into the EcoRI-SmaI site of the pEGFP-C1 vector. Stau262-(318–571) was constructed from Stau262-(243–571)-pEGFP-C1 by cutting out the fragments of HaeIII-BamHI and ligated into the HindIII-BamHI site of the pEGFP-C2 vector in which the HindIII site had been previously cut and blunted. Stau262-(318–412) was constructed from Stau262-(318–571)-pEGFP-C1 by digestion with BamHI and EcoRI, and the larger fragment was blunted and self-ligated. Stau262-(413–571) was constructed from Stau262-(318–571)-pEGFP-C1 by digestion with EcoRI and BspEI, and the larger fragment was blunted and self-ligated. Stau262-(1–105) was constructed from Stau259-pEGFP-C1 by digestion with HpaI and EcoRV, and the larger fragment was blunted and self-ligated. The amino acid substitution mutants were created using the QuikChange site-directed mutagenesis kit (Stratagene). Stau262RBD3* and Stau259RBD3* were constructed from Stau262-pEGFP-C1 and Stau259-pEGFP-C1, respectively, using the following primers: forward, 5′-CCACATATGAAGAGCGCTGTTACCCGGGTGTC-3′, and reverse, 5′-GACACCCGGGTAACAGCGCTCTTCATATGTGG-3′. Stau259L2A, Stau259I4A, and Stau259-L12A were constructed from Stau259-pEGFP-C1 using the following primers: forward, 5′-GCCCGGGATCCATGGCTCAGATAAATCAGAT-G-3′, and reverse, 5′-CATCTGATTTATCTGAGCCATGGATCCCGGGC-3′; forward, 5′-GGGATCCATGCTTCAGGCAAATCAGATGTTTTCG3′, and reverse, 5′-CGAAAACATCTGATTTGCCTGAAGCATGGATCCC-3′; and forward, 5′-GATGTTTTCGGTGCAGGCGAGTCTGGGTGAGCAG-3′, and reverse, 5′-CTGCTCACCCAGACTCGCCTGCACCGAAAACATC-3′, respectively. INQMFSVQLSL was constructed by annealing synthetic oligonucleotides 5′-AATTCATAAATCAGATGTTTTCGGTGCAGCTGAGTCTGG-3′ and 5′-GATCCCAGACTCAGCTGCACCGAAAACATCTGATTTATG-3′ and ligated into the EcoRI-BamHI site of the pEGFP-C1 vector. Cell Cultures and Transfection—HeLa cells were incubated in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% heat-inactivated fetal bovine serum at 37 °C in a 5% CO2 atmosphere. Expression vectors were transfected using the Effectene transfection reagent (Qiagen) using the protocol recommended by the supplier. Leptomycin B was added to medium to a final concentration of 10 nm. Primary Culture of Neurons and Transfection—The hippocampus was dissected from embryonic day 18 Sprague-Dawley rats and digested with 0.25% trypsin in Hanks' buffered salt solution. Neurons were suspended in neurobasal medium (Invitrogen) supplemented with B27 and 0.5 mm l-glutamine and then plated at 3.0 × 105/35-mm plate on polyethylenimine-coated tissue culture dishes at 37 °C in a 5% CO2 atmosphere. On day 10 after plating, neurons were transfected with expression vectors using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. At 24 h after transfection, neurons were fixed with 4% paraformaldehyde in PBS for 15 min at room temperature and then permeabilized with 0.2% Triton X-100 in PBS for 10 min at room temperature. The neurons were blocked with 3% skim milk in PBS for 1 h at room temperature followed by the same solution containing mouse monoclonal anti-MAP2 antibody (Sigma) for 1 h at room temperature. After washing three times with PBS, the neurons were incubated with Alexa 568-labeled goat anti-mouse IgG (Molecular Probes) in the blocking solution for 1 h at room temperature. The neurons were then washed five times with PBS and counterstained with 1 μg/ml Hoechst 33342. RNA Interference—Short interfering RNA for depletion of CRM1 (siCRM1) was prepared as described previously (see also Ref. 17Lund E. Guttinger S. Calado A. Dahlberg J.E. Kutay U. Science. 2004; 303: 95-98Crossref PubMed Scopus (2016) Google Scholar). Sense, UGUGGUGAAUUGCUUAUACd(TT), and antisense, GUAUAAGCAAUUCACCACAd(TT), were obtained from Takara. Transfection of HeLa cells with siCRM1 or mock (transfection reagents only) was performed in 60-mm dishes using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. The cells were harvested at 48 h after transfection and lysed. Then Western blot analysis was performed using a rabbit polyclonal anti-CRM1 antibody (Santa Cruz Biotechnology) or a mouse monoclonal anti-GAPDH antibody (Ambion). Fluorescence Microscopy—All living and fixed cells were examined using a Zeiss Axiovert 200 fluorescent microscope (Carl Zeiss). Stau2 Shuttles between the Nucleus and Cytoplasm—To better understand the dynamic behavior of Stau2 in cells, we initially investigated the subcellular localization of full-length Stau262 by transiently expressing a GFP fusion construct. As shown in Fig. 1, the full-length Stau262 was located primarily in the cytoplasm. However, when a variety of Stau262 deletion mutant constructs, fused to GFP, were transiently expressed in HeLa cells, we found that some mutants such as Stau262-(243–317) and Stau262-(273–373) were clearly localized in the nucleus (Fig. 1), indicating the possibility that Stau262 has the capability of entering the nucleus. Furthermore, the nuclear accumulation of both the Stau262-(243–317) and Stau262-(273–373) mutants suggests that the overlapping domain between RBD3 and RBD4 may mediate nuclear localization. In addition, the localization of Stau262-(318–571), Stau262-(318–412), and Stau262-(413–571) suggests that the domain just following RBD4 may also be involved in nuclear localization. To exclude the possibility that these mutants might have passively diffused into the nucleus followed by the retention, they were fused to MBP (maltose-binding protein)-MBP-GFP (2×MBP-GFP). As shown in Fig. 2, 2×MBP-GFP-Stau262-(243–317) was localized predominantly in the nucleus, indicating that Stau262-(243–317) contains a nuclear localization signal. In contrast, 2×MBP-GFP-Stau262-(318–412) remained in the cytoplasm. These results indicate that the domain between RBD3 and RBD4 mediates the nuclear import of Stau2 but not the downstream domain of the RBD4. On the other hand, the mutant Stau262-(208–272), which is equivalent to the RBD3, was predominantly localized in the cytoplasm, indicating that the RBD3 domain mediates the nuclear export of Stau262. These results indicate that Stau262 is capable of shuttling between the nucleus and cytoplasm. Mutation of RBD3 Prevents the Nuclear Export of Stau262— To confirm that the RBD3 actually mediates the nuclear export of Stau262, we introduced a specific point mutation into the RBD3 that results in a reduction in its RNA binding activity. Based on previous findings on the structure of the double-stranded RNA-binding domain (14Ryter J.M. Schultz S.C. EMBO J. 1998; 17: 7505-7513Crossref PubMed Scopus (392) Google Scholar, 15Ramos A. Grunert S. Adams J. Micklem D.R. Proctor M.R. Freund S. Bycroft M. St. Johnston D. Varani G. EMBO J. 2000; 19: 997-1009Crossref PubMed Scopus (290) Google Scholar), an RBD3 mutant of Stau262 (Stau262RBD3*) was created by replacing phenylalanine 239 with alanine, which has been reported to disrupt the precise folding of the domain (Fig. 3A). HeLa cells were transfected with GFP-Stau262RBD3*, and its subcellular localization was examined 20–24 h after transfection. As shown in Fig. 3B, the full-length Stau262 was localized predominantly in the cytoplasm. In contrast, Stau262RBD3* accumulated in the nucleus, indicating that Stau262 is exported from the nucleus via the RNA binding activity of the RBD3. To address whether this export is dependent on CRM1 (exportin 1), we treated HeLa cells transiently expressing GFP-Stau262 with leptomycin B (LMB), a specific inhibitor of the CRM1-dependent protein export (16Kudo N. Matsumori N. Taoka H. Fujiwara D. Schreiner E.P. Wolff B. Yoshida M. Horinouchi S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9112-9117Crossref PubMed Scopus (834) Google Scholar). No nuclear accumulation of Stau262 was observed (Fig. 3C), indicating that CRM1 is not involved in the RBD3-mediated nuclear export of Stau262. Stau259 Is Exported from the Nucleus by Two Distinct Pathways—To determine whether Stau259, the shorter isoform of Stau2, behaves like Stau262, we examined its subcellular localization by transiently expressing the GFP-fused Stau259 in HeLa cells. As shown in Fig. 4A, like the full-length Stau262, Stau259 was also localized predominantly in the cytoplasm. Therefore, to test whether the nuclear export of Stau259 is mediated by RBD3, we created the RBD3-mutated Stau259 (Stau259RBD3*). Since the secondary structures of Stau262 and Stau259 RBD3 are identical, the mutation was introduced in the same way as that for Stau262. HeLa cells were transfected with GFP-Stau259RBD3*, and its localization was observed (Fig. 4A). Unexpectedly, in contrast to Stau262RBD3*, Stau259RBD3* showed no nuclear accumulation. From these findings, we speculate that Stau259 is exported via an alternate export pathway, for example, a CRM1-dependent one. Therefore, HeLa cells transfected with GFP-Stau259RBD3* were treated with LMB. As shown in Fig. 4B, LMB treatment resulted in the nuclear accumulation of Stau259RBD3*. In contrast, wild type full-length Stau259 was localized predominantly in the cytoplasm, indicating that the RBD3-mediated nuclear export pathway, which is not sensitive to LMB, is also functional for Stau259. Thus, Stau259 appears to be exported from the nucleus by two distinct pathways; one is mediated by the RBD3, which is CRM1-independent, and the other is CRM1-dependent. To confirm this, we performed RNA interference targeting CRM1. A short interfering RNA duplex targeted to residues 90–108 of the human CRM1 open reading frame (siCRM1) was prepared according to the previous report (17Lund E. Guttinger S. Calado A. Dahlberg J.E. Kutay U. Science. 2004; 303: 95-98Crossref PubMed Scopus (2016) Google Scholar) and transfected to HeLa cells. At 48 h after transfection, the cells were harvested, and the expression level of endogenous CRM1 was analyzed by Western blotting. As shown in Fig. 4C, this treatment specifically reduced the expression level of CRM1. To test the effect of the down-regulation of CRM1 on the cellular localization of Stau259, HeLa cells were transfected with siCRM1 together with GFP-Stau259 or GFP-Stau259RBD3*. As shown in Fig. 4D, whereas the down-regulation of CRM1 resulted in the predominant nuclear localization of GFP-Stau259RBD3*, GFP-Stau259 was primarily localized in the cytoplasm, which was consistent with the results obtained by using LMB. Identification of a CRM1-dependent Nuclear Export Signal of Stau259 at the N-terminal Domain—Since the primary structure of Stau259 is different from that of Stau262 only in the N-terminal domain (Fig. 5A), we hypothesized that the N-terminal domain of Stau259 mediates the CRM1-dependent nuclear export. For this, the deletion mutant Stau259-(1–105) was constructed as a GFP fusion protein and transfected into HeLa cells. As shown in Fig. 5B, GFP-Stau259-(1–105) was localized predominantly in the cytoplasm. In addition, LMB treatment promoted the nuclear localization of GFP-Stau259-(1–105). On the other hand, the localization of GFP-Stau262-(1–137) was similar to that of GFP alone and was not affected by LMB treatment. These results indicate that the N-terminal domain of Stau259 mediates the CRM1-dependent nuclear export. It is well known that nuclear export signals (NESs) that are recognized by CRM1 are frequently composed of a stretch of characteristically spaced hydrophobic amino acids such as leucine and isoleucine, as was first reported for the human immunodeficiency virus type 1 Rev (18Fischer U. Huber J. Boelens W.C. Mattaj I.W. Luhrmann R. Cell. 1995; 82: 475-483Abstract Full Text PDF PubMed Scopus (976) Google Scholar) and protein kinase inhibitor (19Wen W. Meinkoth J.L. Tsien R.Y. Taylor S.S. Cell. 1995; 82: 463-473Abstract Full Text PDF PubMed Scopus (991) Google Scholar) proteins. To test whether the hydrophobic amino acid-rich sequence INQMFSVQLSL in the N-terminal of Stau259 functions as the NES that is recognized by CRM1, HeLa cells were transfected with GFP-INQMFSVQLSL, and its localization was examined (Fig. 6A). GFP-INQMFSVQLSL was localized predominantly in the cytoplasm, and LMB treatment led to its nuclear localization, indicating that the sequence INQMFSVQLSL is recognized by CRM1. To confirm that INQMFSVQLSL functions as the CRM1-dependent NES of Stau259, two point mutants of Stau259RBD3* were first constructed by replacing isoleucine 4 and leucine 12 with alanine, respectively, because Stau259RBD3* is convenient for assessing the CRM1-dependent pathway. HeLa cells were transfected with GFP-Stau259RBD3*/I4A, GFP-Stau259RBD3*/L12A, or GFP-Stau259RBD3*/L2A as a control (Fig. 6B). Mutations I4A and L12A abolished the CRM1-dependent nuclear export activity of GFP-Stau259RBD3*, although L2A mutation did not. To test whether these point mutations influence the localization of Stau259, which is also exported by the RBD3-dependent export pathway, GFP-Stau259I4A and GFP-Stau259L12A were transfected into HeLa cells (Fig. 6C). As in the case in which HeLa cells transiently expressing GFP-Stau259 were treated with LMB, GFP-Stau259I4A and GFP-Stau259L12A were localized predominantly in the cytoplasm, which confirms that Stau259 is also exported via a CRM1-independent pathway. Stau2 Export Mutants Show the Same Localization in Hippocampal Neurons as in HeLa Cells—It is known that Stau2 is mainly expressed in the brain and is involved in the dendritic transport of mRNA in neurons (7Duchaine T.F. Hemraj I. Furic L. Deitinghoff A. Kiebler M.A. DesGroseillers L. J. Cell Sci. 2002; 115: 3285-3295PubMed Google Scholar). To test whether the same nuclear export pathways for Stau2 exist in neurons as in HeLa cells, rat hippocampal neurons were transfected with GFP-fused constructs of Stau2 export mutants (Fig. 7). Although GFP-Stau262 was localized predominantly in the somatodendritic domain, GFP-Stau262RBD3* accumulated in the nucleus, indicating that Stau262 is, in fact, exported from the nucleus only in an RBD3-mediated manner in neurons. Furthermore, Stau259 export mutants showed a subcellular localization in neurons that was similar to that in HeLa cells. That is, GFP-Stau259RBD3* and GFP-Stau259I4A were predominantly localized in the cytoplasm in a similar fashion as GFP-Stau259, whereas GFP-Stau262RBD3*/I4A accumulated in the nucleus. In addition, LMB treatment of neurons resulted in the nuclear accumulation of GFP-Stau259RBD3* (data not shown). These results indicate that Stau259 is exported from the nucleus via two distinct pathways, an RBD3-mediated one and a CRM1-dependent one in neurons, as in HeLa cells. Using Stau2 deletion mutant constructs, we report here that Stau2 is a nucleocytoplasmic shuttle protein. In addition, the findings show that the region between RBD3 and RBD4 functions as the nuclear localization signal of Stau2, consistent with the recent report by Macchi et al. (20Macchi P. Brownawell A.M. Grunewald B. DesGroseillers L. Macara I.G. Kiebler M.A. J. Biol. Chem. 2004; 279: 31440-31444Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), although the import factor that recognizes the nuclear localization signal remains to be determined. We next analyzed the nuclear export of Stau2 in more detail. First, we showed that the nuclear export of Stau262 is mediated by RBD3. This is consistent with a recent report by Macchi et al. (20Macchi P. Brownawell A.M. Grunewald B. DesGroseillers L. Macara I.G. Kiebler M.A. J. Biol. Chem. 2004; 279: 31440-31444Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), who demonstrated that the RBD3 of Stau262 interacts with exportin 5 in an RNA-dependent manner in vitro. Moreover, they showed that the down-regulation of exportin 5 by RNA interference resulted in the nuclear accumulation of Stau262, indicating that exportin 5 is the nuclear export factor of Stau262. Furthermore, Brownawell and Macara (21Brownawell A.M. Macara I.G. J. Cell Biol. 2002; 156: 53-64Crossref PubMed Scopus (133) Google Scholar) reported that the RBD of ILF3, Spnr, Staufen1, and protein kinase R bind to exportin 5, suggesting that exportin 5 participates in the nuclear export of dsRNA-binding proteins. Interestingly, we report, for the first time, that a functional difference exists among the Stau2 isoforms. That is, Stau262 is exported from the nucleus only in a RBD3-mediated manner, whereas Stau259 is exported by two distinct pathways, a RBD-mediated one and a CRM1-dependent one. In addition, we identified the NES sequence at the N-terminal end of Stau259, indicating that alternative splicing creates a new functional domain for nuclear export. It is reasonable to speculate that another Stau2 isoform, Stau252, is exported by the same two pathways because Stau252 contains the same RBD3 and the same NES sequence recognized by CRM1 as Stau259. The question arises as to the nature of the biological significance of the export of Stau259 from the nucleus in a CRM1-dependent manner. Exportin 5 preferentially recognizes RNAs containing a minihelix motif, a structure that comprises a double-stranded stem of over 14 nucleotides with a base-paired 5′ end and a 3–8-nucleotide protruding 3′ end (22Gwizdek C. Ossareh Nazari B. Brownawell A.M. Doglio A. Bertrand E. Macara I.G. Dargemont C. J. Biol. Chem. 2003; 278: 5505-5508Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 23Gwizdek C. Ossareh Nazari B. Brownawell A.M. Evers S. Macara I.G. Dargemont C. J. Biol. Chem. 2004; 279: 884-891Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). The RBD3 of Stau2 is a major dsRNA binding determinant (7Duchaine T.F. Hemraj I. Furic L. Deitinghoff A. Kiebler M.A. DesGroseillers L. J. Cell Sci. 2002; 115: 3285-3295PubMed Google Scholar). Therefore, when Stau262 is exported from the nucleus by exportin 5, Stau262 appears to bind to and, therefore, to carry restricted minihelix-RNAs from the nucleus. On the other hand, it is likely that Stau259 as well as Stau252, which are exported by CRM1, are able to transport other sets of dsRNAs that are unable to bind to exportin 5 or bind to the RBD3 in the absence of exportin 5. Considering this, along with the data that the expression of Stau259 is more abundant than Stau262 in the brain of adult rodents (7Duchaine T.F. Hemraj I. Furic L. Deitinghoff A. Kiebler M.A. DesGroseillers L. J. Cell Sci. 2002; 115: 3285-3295PubMed Google Scholar, 11Mallardo M. Deitinghoff A. Muller J. Goetze B. Macchi P. Peters C. Kiebler M.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2100-2105Crossref PubMed Scopus (140) Google Scholar), we propose the possibility that the CRM1-dependent nuclear export of Stau259 plays an important role in transporting various RNAs out of the nucleus. However, the possibility that Stau2 may be exported from the nucleus without binding any RNA cannot be excluded. Kress et al. (24Kress T.L. Yoon Y.J. Mowry K.L. J. Cell Biol. 2004; 165: 203-211Crossref PubMed Scopus (103) Google Scholar) proposed a model for the vegetal RNA localization pathway in xenopus oocytes in which xenopus Staufen is recruited into the core ribonucleoprotein complex in the cytoplasm that has been assembled in the nucleus. Further study will be required to determine whether Stau2 must be imported into the nucleus once to enter RNA granules." @default.
• W2064848035 created "2016-06-24" @default.
• W2064848035 creator A5058319582 @default.
• W2064848035 creator A5084921992 @default.
• W2064848035 date "2004-11-01" @default.
• W2064848035 modified "2023-10-15" @default.
• W2064848035 title "Alternative Splicing of Staufen2 Creates the Nuclear Export Signal for CRM1 (Exportin 1)" @default.
• W2064848035 cites W1566430503 @default.
• W2064848035 cites W1636792448 @default.
• W2064848035 cites W1963705823 @default.
• W2064848035 cites W1964350517 @default.
• W2064848035 cites W1989712305 @default.
• W2064848035 cites W2008922773 @default.
• W2064848035 cites W2009747268 @default.
• W2064848035 cites W2016153572 @default.
• W2064848035 cites W2029842408 @default.
• W2064848035 cites W2031408226 @default.
• W2064848035 cites W2048716839 @default.
• W2064848035 cites W2052595458 @default.
• W2064848035 cites W2062477253 @default.
• W2064848035 cites W2076347860 @default.
• W2064848035 cites W2080018502 @default.
• W2064848035 cites W2080559673 @default.
• W2064848035 cites W2080924644 @default.
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