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Vol. 19, Issue 7, 3008-3019, July 2008
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*Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institute, Stockholm SE-171 77, Sweden; Department of Neuroscience, Karolinska Institute, Stockholm SE-171 77, Sweden; Serveis Cientificotècnics, Universitat de Barcelona, E-08028 Barcelona, Spain; and Department of Molecular Biology and Functional Genomics, Stockholm University, Stockholm, SE-10691, Sweden
Submitted October 26, 2007;
Revised April 8, 2008;
Accepted May 6, 2008
Monitoring Editor: A. Gregory Matera
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2002). In mammals, there are more than 20 major and a large number of minor hnRNP species, designated A1 to U hnRNPs (Dreyfuss et al., 1993, 2002; Krecic and Swanson, 1999). Within this large protein family, hnRNPs of the A/B type exhibit a well-defined modular structure with two conserved tandemly repeated RNA-binding domains (RBDs), and a divergent C-terminal region termed auxiliary domain. Certain hnRNPs of the A/B-type are in complex with actin (Percipalle et al., 2002). These proteins included the hnRNP A/B protein or CBF-A (CArG-box binding factor A; Bemark et al., 1998), hnRNP A2, hnRNP A3 as well as hnRNP U (Kiledjian and Dreyfuss, 1992). Specific actin-hnRNP interactions were shown to be essential for RNA polymerase II transcription both in insect and mammalian cells, which indicates a key role for hnRNPs in mRNA synthesis (Percipalle and Visa, 2006). In addition, consistent with the idea that shuttling hnRNPs accompany mRNA transcripts from gene to polysomes, a number of other specialized functions have been ascribed to the A/B-type hnRNPs, including a role in mRNP export, cytoplasmic transport, and localization (Dreyfuss et al., 2002).
In the cytoplasm, mRNA transport and localization have been studied in Drosophila melanogaster, Xenopus laevis oocytes, yeast and, more recently, in mammalian somatic cells (Palacios and St. Johnson, 2001; Shav-Tal and Singer, 2005). cis-acting sequences in the RNA and cellular transacting factors mediate cytoplasmic transport and localization of many eukaryotic mRNAs, including β-actin mRNA in fibroblasts (Ross et al., 1997; Hüttelmaier et al., 2005), Vg1 mRNA in X. laevis oocytes (Deshler et al., 1997) and myelin basic protein (MBP) mRNA in oligodendrocytes (Ainger et al., 1993, 1997). For transport of MBP mRNP granules to the myelin compartment, a 21-nucleotide sequence in the 3'-untranslated region (UTR), known as RTS (RNA trafficking sequence), works as cis-acting sequence and is necessary and sufficient for MBP mRNA transport (Ainger et al., 1997). In vitro, the RTS is recognized by the transacting factors hnRNP A2 and A3, which are presumably part of transported granules. In oligodendrocytes, hnRNP A2 was found to facilitate transport of microinjected RTS-containing transcripts and a similar role in RTS-mediated mRNA trafficking was suggested for hnRNP A3 in hippocampal neurons (Hoeck et al., 1998; Carson et al., 2001; Ma et al., 2002; Smith, 2004; Carson and Barbarese, 2005).
MBP, together with the proteolipid protein (PLP) and its isoform DM20 are major constituents of purified myelin, and they are required to stabilize the apposed myelin membranes in compact myelin (Gielen et al., 2004). The molecular mechanisms underlying the regulation of MBP biogenesis are still unclear. In an attempt to identify transacting factors associated with transported granules, recent proteomic studies showed that hnRNP U and CBF-A are among the hnRNPs that are found in RNP granules isolated from developing and adult mouse brains (Kanai et al., 2004; Elvira et al., 2006). CBF-A is a shuttling hnRNP component of pre-mRNP/mRNP particles (Percipalle et al., 2002). However, despite of the above observations, at this stage, its potential role in cytoplasmic mRNA transport and MBP biogenesis has not yet been investigated.
In this study, we show evidence that CBF-A binds the MBP mRNA RTS sequence and is required in vivo for MBP mRNA transport in oligodendrocytes. Based on these observations, we propose a general role for CBF-A as a cellular transacting factor in cytoplasmic RNA transport and localization.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Culture
HeLa and oli-neu cells (gift from J. Trotter, University of Mainz) were maintained as previously shown (Jung et al., 1995; Percipalle et al., 2002). Briefly, oli-neu cells were propagated in Sato medium containing 1% horse serum and differentiated for 7–10 d with dibutyryl cAMP (Sigma-Aldrich, St. Louis, MO), as described (Jung et al., 1995).
Immunohistochemistry on Mouse Forebrain Sections
Adult C57bl6 mice (Charles River, Wilmington, MA) were transcardially perfused with PBS followed by 4% formaldehyde in PBS, and brains were dissected and postfixed overnight at 4°C. Coronal sections were made at 35 µm using a vibratom (Leica, Heidelberg, Germany). Immunostaining for CBF-A, MBP, and CNPase was performed on free-floating sections (see Supplementary Information).
Primary Cells
Adult mouse neural stem cells were prepared and cultured as neurospheres and differentiated as described (Rietze and Reynolds, 2006), with minor modifications (see Supplementary Information for a detailed description of the protocol used). Fetal rat neural stem cells were prepared as described in the Supplementary Information.
Immunofluorescence Microscopy, Immuno-FISH, and Gene Silencing
Immunofluorescence and immuno-FISH on cells grown on coverslips were already described (Singer and Ward, 1982; Percipalle et al., 2002). For in situ detection of MBP mRNA, the RNA probe 5'-CCAUGCUCUCUGGCUCCUUGGCGGUGUGCCUGUCU-3' was digoxigenin-labeled in the 5' end. Confocal images were collected using a Zeiss LSM 510 META confocal laser scanning microscope (Jena, Germany; Percipalle et al., 2002).
For posttranscriptional silencing of the CBF-A gene, the siGENOME SMART pool duplex against the mouse HNRPAB gene (NM_010448 [GenBank] ) or the control ECFP (enhanced cyan fluorescent protein) gene were transfected into oli-neu cells according to the manufacturer's instructions (Dharmacon Research, Boulder, CO). Sequences can be found in Supplementary Information.
Cloning, Expression, and Protein Purification
Full-length hnRNP A2 (forward primer 5'-GGAATTCTTAGCGACTGAGTCCGCGATG, reverse primer 5'-ATAAGAATGCGGCCGCTGAAGCTGTTCTGTTACCTCTG) and hnRNP A3 (Ma et al., 2002) were cloned in pGEM-T (Promega, Madison, WI) and subsequently in pET30a (+) for expression (Novagen, Madison, WI). Constructs were verified by DNA sequencing. Full-length glutathione S-transferase (GST)-tagged CBF-A (gift of Tomas Leanderson, Lund University) was expressed from a pGEX plasmid vector (see Supplementary Information).
Protein–Protein Interaction Assays
For pulldown experiments, recombinant hnRNP A2, hnRNP A3, or CBF-A constructs were coupled to protein S agarose or glutathione beads (Novagen; GE Healthcare, Waukesha, WI) and incubated with HeLa nuclear extracts (see Supplementary Information).
Protein and RNA Immunoprecipitation
For protein immunoprecipitations, total protein extracts prepared from oli-neu cells were incubated with the anti-CBF-A antibody, with the anti-HA tag antibody, and with nonspecific mouse IgGs. Where indicated, extracts were incubated with RNase A (see Supplementary Information). The immunoprecipitated fractions from untreated and RNase A–treated extracts were resolved by SDS-PAGE and analyzed on immunoblots with antibodies against CBF-A and hnRNP A2. For analysis of the RNA species associated with CBF-A, the RNA was extracted from the fractions immunoprecipitated with the anti-CBF-A antibody or mock and IgG control immunoprecipitation assays using the Trizol reagent, followed by DNase I (Invitrogen, Carlsbad, CA) treatment and subsequently made into cDNA by Superscript II (Invitrogen) using random primers. The samples were then analyzed by quantitative RT-PCR (qRT-PCR) with primers specific to MBP mRNA exon 1, MBP mRNA exon 2, and Sox 10 and Sox9 mRNA (see Supplementary Information for primer sequences and qRT PCR analysis).
Protein–RNA Interaction Assays
RNA affinity chromatography was performed as described (Hoek et al., 1998). For each RNA-binding experiment, 500 pmol of 5'-biotinylated wild-type RTS (wtRTS; 5'-AGACAGGCACACCGCCAAGGAGCCAGAGAGCAUGG-3') and scrambled RTS (scrRTS; 5'-GGGAGCGGAGAAACAAGCACCGAACCCGCAACUGG-3') were coupled to streptavidin-coated Sepharose (GE Healthcare) and incubated with nuclear, cytoplasmic, and high-salt extracts. Bound proteins were resolved by SDS-PAGE and analyzed on immunoblots (see Supplementary Information).
For electrophoretic mobility shift assay (EMSA), wtRTS, and scrRTS were 5' end-labeled using 
-33P-ATP (GE Healthcare) and T4 polynucleotide kinase (New England Biolabs, Beverly, MA). Purified hnRNP A2, hnRNP A3, and CBF-A were incubated with 
50 fmol of 33P-labeled wtRTS or scrRTS in EMSA buffer (20 mM HEPES, pH 7.6, 5 mM MgCl2, 40 mM KCl, 1 mM DTT, 5% glycerol) containing heparin (5 µg/µl) and BSA (100 µg/ml) for 30 min on ice. Protein–RNA complexes were resolved by native gel electrophoresis and analyzed with a Fuji-BAS 2000 phosphorimager (Tokyo, Japan).
Immunoelectron Microscopy
After fixation, the cells were either cryosectioned or cryosubstituted and embedded in Lowicryl K4M (Polysciences, Warrington, PA; see Supplementary Information). Cryosectioned samples provided a good definition of nuclear and membrane structures, whereas cryosubstitution offered a superior preservation of the cytoskeleton. For immunolabeling, binding of the anti-CBF-A antibody was detected with either a secondary antibody or protein A coupled to 10-nm diameter colloidal gold particles. Sections were observed in a JEM-1010 electron microscope (Jeol, Tokyo, Japan), and photographic negatives were scanned with an Artixcan 2500f scanner (Microtek International, Hsinchu, Taiwan; see Supplementary Online Information).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Evidence that CBF-A is a component of pre-mRNP/RNP particles prompted us to determine whether CBF-A is in a multiprotein complex together with core hnRNP proteins such as hnRNP A2 and hnRNP A3. We first fractionated nuclear proteins prepared from HeLa cells by ultracentrifugation on a sucrose gradient and monitored coelution of CBF-A on immunoblots, using a novel affinity-purified peptide-specific anti-CBF-A antibody that recognizes the larger p42 isoform (Figure 1A, cf. lane 3). CBF-A was found to coelute with hnRNP A2/A3 in an RNA-dependent manner (Figure 1B). To demonstrate the suggested association of CBF-A with hnRNP A2/A3, tagged CBF-A, hnRNP A2, and hnRNP A3 full-length constructs were recombinantly expressed and affinity-purified for in vitro protein–protein interactions (Figure 1C). GST-tagged CBF-A and S-tagged hnRNP A2 and A3 were coupled to either glutathione beads or protein S beads and incubated with HeLa nuclear protein extracts. Coprecipitated proteins were resolved by SDS-PAGE and monitored on immunoblots. Figure 1E shows that recombinant CBF-A, hnRNP A2, and hnRNP A3 reciprocally coprecipitated the endogenous proteins, as well as hnRNP U, a newly identified protein component of transported RNA granules (Elvira et al., 2006; Kanai et al., 2004). None of the proteins could be coprecipitated from nuclear extracts using protein S beads or GST beads, further supporting the specificity of the reaction (Figure 1D, cf. lanes 3and 10, and Figure 1E). Overall, these results are consistent with the view that subcellular fractions of CBF-A, hnRNP A2, hnRNP A3, and hnRNP U are physically associated and are contained in a nuclear multiprotein complex.
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In Oligodendrocytes CBF-A Associates with MBP mRNA Granules
If CBF-A specifically binds RTS sequences found within the MBP mRNA, it is possible that in oligodendrocytes CBF-A remains associated with transported mRNA granules outside the cell nucleus. Consistently, immunoblots of protein extracts prepared from different mouse tissues revealed that CBF-A is ubiquitously expressed (Figure 2E) and that the anti-CBF-A antibody is monospecific in brain tissue (Figure 2E, lane 2).
On the basis of the above, we analyzed the in vivo distribution of CBF-A in the myelin-rich regions present in the forebrain of adult mice. In agreement with its ubiquitous expression, immunostaining of mouse brain sections with the anti-CBF-A antibody followed by confocal microscopy revealed that cellular CBF-A is expressed all over in the forebrain, showing distinctive nuclear localization (Figure 3). Interestingly, a fraction of CBF-A was also found outside the cell nucleus in discrete particles, reminiscent of transported granules in oligodendrocytes, with different sizes and signal intensities depending on the brain regions analyzed (Figure 3, cf. E and M). To test whether in oligodendrocytes CBF-A displays a granular distribution, we performed triple-immunostaining of brain sections with the anti-CBF-A antibody, a rat mAb against MBP, which specifically labels myelin fibers, and a mouse mAb against 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase), which labels oligodendrocytes membranes and is commonly used as marker for oligodendrocytes. Remarkably, in nuclei encapsulated by CNPase we detected very little CBF-A, whereas most of the CBF-A–positive granules were found in close proximity to CNPase and/or MBP, suggesting a specialized cytoplasmic function for CBF-A, presumably in processes emanating from the cell body (Figure 3, cf. I and Q). Consistent with the in vivo distribution detected in mouse forebrain, CBF-A–positive granules were also revealed in primary oligodendrocytes, astrocytes, and neurons obtained by in vitro differentiation of fetal rat and adult mouse neural stem cells (Figure 4 and Supplementary Figure S1). In all these cases, CBF-A–positive granules were revealed along the microtubule-rich processes.
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). Differentiated oli-neu cells were subjected to immunofluorescence in situ hybridization (immuno-FISH) experiments with the anti-CBF-A antibody and a specific 5' digoxigenin end-labeled RNA probe hybridizing with the endogenous MBP mRNA RTS. Confocal microscopy revealed that CBF-A–positive granules distributed along oligodendrocytes processes partly colocalize with MBP mRNA in granular structures (Figure 5, A–H, see arrowheads in H), similar to hnRNP A2 (Figure 5, I–P, see arrows in P). In support of a colocalization between endogenous CBF-A and RTS-containing MBP mRNA, we next performed unbiased statistical analysis on the fluorescence intensity levels derived from the corresponding confocal images and as previously described (Ma et al., 2002). The results revealed linear correlations between the CBF-A and RTS signals (Figure 5B). Quantification of the number of colocalization events occurring in individual particles showed that roughly 88% of the granules contains CBF-A and RTS-containing mRNA. As expected, a similar correlation was revealed between the hnRNP A2 and RTS signals (Figure 5C), where 90% of the granules contained both hnRNP A2 and RTS-containing mRNA. Furthermore, double immunostaining experiments demonstrated a linear correlation also between the granular distributions of CBF-A and hnRNP A2 (Figure 6, A and B), and CBF-A was found to colocalize with 
-tubulin along oli-neu processes (Supplementary Figure 2). Finally triple oli-neu immunofluorescence staining for the MBP mRNA RTS, hnRNP A2, and CBF-A showed that more than 80% of endogenous MBP mRNA granules were positively stained for RTS RNA, CBF-A, and hnRNP A2 (Supplementary Figure S3). We conclude that CBF-A binds RTS sequences and is present in MBP mRNA-containing granules.
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). As can be seen, the bars diagram in Figure 7 shows a significant enrichment (5.9% of the input) of exon1-containing MBP mRNA transcripts (Figure 7B), an exon that is present in all MBP isoforms (Boggs, 2006). A considerable enrichment was also observed (7.4% of the input) for the MBP mRNA isoforms that contain exon 2 (Figure 7B), an exon that is mainly expressed in the early stages of myelination (Boggs, 2006). Finally, MBP mRNA enrichment was not revealed in the control immunoprecipitated fractions (Figure 7B). We conclude that in differentiating oli-neu cells a considerable proportion of CBF-A is specifically associated with the MBP mRNA, presumably as part of MBP mRNA granules.
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; Barbarese et al., 1995). Both large and small granules are found in association with the cytoskeleton. Finally, the immunoelectron microscopy experiment also revealed that CBF-A reaches the myelin compartment. Indeed, significant labeling was observed in association with the concentric multilamellar formations that are characteristic of the differentiating oligodendrocytes (Figure 9, E, G, and H).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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CBF-A and hnRNP A2 are both present in MBP mRNA granules, and they are both required for MBP mRNA transport and localization. These observations raise the question as to why the same mRNA granule requires the presence of at least two transacting factors. At this stage it is rather difficult to address this issue at the molecular level. However, the linear correlations on the fluorescence signals observed between CBF-A and MBP mRNA, between hnRNP A2 and MBP mRNA as well as between CBF-A and hnRNP A2, and the fact that CBF-A and hnRNP A2 can be coimmunoprecipitated in an RNA-dependent manner overall suggest that CBF-A and hnRNP A2 are simultaneously present in the same MBP mRNP complex. If this is the case, CBF-A and hnRNP A2 may cooperate for MBP mRNA transport and localization. Their roles could be dependent on the local environment and the set of interactions required for the establishment of trafficking intermediates, for instance, to determine polarity during RNA trafficking (Carson and Barbarese, 2005).
CBF-A is ubiquitously expressed and has been suggested to have a role in transcriptional control (Kamada and Miwa, 1992; Bemark et al., 1998; Leverrier et al., 2000; Mikheev et al., 2000). Because it is also a component of shuttling pre-mRNP/mRNP particles (Percipalle et al., 2002), our results suggest that CBF-A associates with mRNAs in the cell nucleus and accompanies its target mRNAs to their cytoplasmic destination. In support of this hypothesis the immunoelectron microscopy performed with our anti-CBF-A antibody in oli-neu and HeLa cells showed that CBF-A localizes in the nucleoplasm (Figure 9B and Supplementary Figure S4B). Occasionally, CBF-A was found at or near nuclear pores (Supplementary Figure S4C), which is consistent with the proposal that CBF-A remains associated with the mRNP during nucleo-cytoplasmic transport. In the cytoplasm of oli-neu and HeLa cells, CBF-A was often found in dense granules with a diameter of 30–40 nm (Figure 9F and Supplementary Figure S4, D and E). These CBF-A–containing granules, which were often seen in association with cytoskeletal fibers, are likely to be mRNA transport granules. In either case, our observations underscore the general idea that the cytoplasmic fate of each mRNA is predetermined by the assembly of the mRNP complex in the cell nucleus (Daneholt, 2001).
We revealed CBF-A–positive granules in astrocytes and neurons where mRNA transcripts containing RTS-like sequences were identified by computational approaches (Ainger et al., 1997). Moreover, CBF-A was found associated with the cytoskeleton in HeLa cells. Altogether these observations, strongly suggest that CBF-A has a role in cytoplasmic mRNA transport and its function may not be exclusive of oligodendrocytes.
Given the ubiquitous distribution of CBF-A and its specific role in MBP mRNA transport in oligodendrocytes, we suggest that CBF-A has a general role as a transacting factor in the establishment of asymmetric mRNA and/or protein distributions. CBF-A is likely to act on the localization of multiple mRNAs in HeLa cells as well as in other cell types, the MBP mRNA being a specific case related to a very specialized cellular function.
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ACKNOWLEDGMENTS |
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Footnotes |
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Address correspondence to: Piergiorgio Percipalle (piergiorgio.percipalle{at}ki.se)
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REFERENCES |
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Ainger, K., Avossa, D., Diana, A. S., Barry, C., Barbarese, E., and Carson, J. H. (1997). Transport and localization elements in myelin basic protein mRNA. J. Cell Biol 138, 1077–1087.[CrossRef]
Barbarese, E., Koppel, D. E., Deutscher, M. P., Smith, C. L., Ainger, K., Morgan, F., and Carson, J. H. (1995). Protein translation components are colocalized in granules in oligodendrocytes. J. Cell Sci 108, 2781–2790.[Abstract]
Boggs, J. M. (2006). Myelin basic protein: a multifunctional protein. Cell Mol. Life Sci 63, 1945–1961.[CrossRef][Medline]
Bemark, M., Olsson, H., Heinegård, D., and Leanderson, T. (1998). Purification and characterization of a protein binding to the SP6 k promoter. J. Biol. Chem 273, 18881–18890.
Carson, J. H., Cui, H., Krueger, W., Schlepchenko, B., Brumwell, C., and Barbarese, E. (2001). RNA trafficking in oligodendrocytes. Results Probl. Cell Differ 34, 69–81.[Medline]
Carson, J. H., and Barbarese, E. (2005). Systems analysis of RNA trafficking in neural cells. Biol. Cell 97, 51–62.[CrossRef][Medline]
Daneholt, B. (2001). Assembly and transport of a pre-messenger RNP particle. Proc. Natl. Acad. Sci. USA 98, 7012–7017.
Dean, J.L.E., Sully, G., Wait, R., Rawlinson, L., Clark, A. R., and Saklatvala, J. (2002). Identification of a novel AU-rich-element-binding protein which is related to AUF1. Biochem. J 366, 709–719.[CrossRef][Medline]
Deshler, J. O., Highett, M. I., and Scnapp, B. J. (1997). Localization of Xenopus Vg1 mRNA by Vera protein and the endoplasmic reticulum. Science 276, 1128–1131.
Dreyfuss, G., Matunis, M. J., Pinõl-Roma, S., and Burd, C. G. (1993). hnRNP proteins and the biogenesis of mRNA. Annu. Rev. Biochem 62, 289–321.[CrossRef][Medline]
Dreyfuss, G., Kim, V. N., and Kataoka, N. (2002). Messenger-RNA-binding proteins and the messenger they carry. Nat. Rev. Mol. Cell Biol 3, 195–205.[CrossRef][Medline]
Elvira, G. et al. (2006). Characterization of an RNA granule from developing brain. Mol. Cell Proteomics 5, 635–651.
Gielen, E., Baron, W., Vandeven, M., Steels, P., Hoekstra, D., and Ameloot, M. (2004). Rafts in oligodendrocytes: evidence and structure-function relationship. Glia 54, 499–512.[CrossRef]
Hoek, K. S., Kidd, G. J., Carson, J. H., and Smith, R. (1998). hnRNP A2 selectively binds the cytoplasmic transport sequence of myelin basic protein mRNA. Biochemistry 37, 7021–7029.[CrossRef][Medline]
Hüttelmaier, S., Zenklusen, D., Lederer, M., Dictenberg, J., Lorenz, M., Meng, X., Bassell, G. J., Condeelis, J., and Singer, R. H. (2005). Spatial regulation of beta-actin translation by Src-dependent phosphorylation of ZBP1. Nature 438, 432–435.[CrossRef][Medline]
Jung, M. et al. (1995). Lines of murine oligodendroglial precursor cells immortalized by an activated neu tyrosine kinase show distinct degrees of interaction with axons in vitro and in vivo. Eur. J. Neurosci 7, 1245–1265.[CrossRef][Medline]
Kamada, S., and Miwa, T. (1992). A protein binding to CArG box motifs and to single-stranded DNA functions as transcriptional repressor. Gene 119, 229–236.[CrossRef][Medline]
Kanai, Y., Dohmae, N., and Hirokawa, N. (2004). Kinesin transports RNA: isolation and characterization of an RNA-transporting granule. Neuron 43, 513–525.[CrossRef][Medline]
Kiledjian, M., and Dreyfuss, G. (1992). Primary structure and binding activity of the hnRNP U protein: binding RNA through RGG box. EMBO J 11, 2655–2664.[Medline]
Krecic, A. M., and Swanson, M. S. (1999). hnRNP complexes: composition, structure and function. Curr. Opin. Cell Biol 11, 363–371.[CrossRef][Medline]
Leverrier, S., Cinato, E., Paul, C., Derancourt, J., Bemark, M., Leandersson, T., and Legraverend, C. (2000). Purification and cloning of type A/B hnRNP proteins involved in transcriptional activation from the rat spi2 gene GAGA box. Biol. Chem 381, 1031–1040.[CrossRef][Medline]
Ma, A.S.W., Moran-Jones, K., Shan, J., Munro, T. P., Snee, M. J., Hoek, K. S., and Smith, R. (2002). Heterogeneous nuclear ribonucleoprotein A3, a novel RNA trafficking response element-binding protein. J. Biol. Chem 277, 18010–18020.
Mikheev, A. M., Mikheev, S. A., Zhang, Y., Aebersold, R., and Zarbl, H. (2000). CArG binding factor A (CBF-A) is involved in transcriptional regulation of the rat Ha-ras promoter. Nucleic Acids Res 28, 3762–3770.
Palacios, I., and St. Johnson, D. (2001). Getting the message across: the intracellular localization of mRNAs in higher eukaryotes. Annu. Rev. Cell Dev. Biol 17, 569–614.[CrossRef][Medline]
Percipalle, P., Jonsson, A., Nashchekin, D., Karlsson, C., Bergman, T., Guialis, A., and Daneholt, B. (2002). Nuclear actin is associated with a specific subset of hnRNP A/B-type proteins. Nucleic Acids Res 30, 1725–1734.
Percipalle, P., and Visa, N. (2006). Molecular functions of nuclear actin. J. Cell Biol 172, 967–971.
Rietze, R. L., and Reynolds, B. A. (2006). Neural stem cell isolation and characterization. Methods Enzymol 419, 3–23.[CrossRef][Medline]
Ross, A., Oleynikov, Y., Kislauskis, E., Taneja, K., and Singer, R. (1997). Characterization of a beta actin mRNA zipcode-binding protein. Mol. Cell. Biol 17, 2158–2165.[Abstract]
Shav-Tal, Y., and Singer, R. H. (2005). RNA localization. J. Cell Sci 118, 4077–4081.
Singer, R. H., and Ward, D. C. (1982). Actin gene expression visualized in chicken muscle tissue culture by using in situ hybridization with a biotinylated nucleotide analog. Proc. Natl. Acad. Sci. USA 79, 7331–7335.
Smith, R. (2004). Moving molecules: mRNA trafficking in Mammalian oligodendrocytes and neurons. Neuroscientist 10, 495–500.
Wegner, M., and Stolt, C. C. (2005). From stem cells to neurons and glia: a Soxist's view of neural development. Trends Neurosci 28, 583–588.[CrossRef][Medline]
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