Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M706082200 on January 24, 2008

J. Biol. Chem., Vol. 283, Issue 12, 7531-7541, March 21, 2008
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
283/12/7531    most recent
M706082200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ratti, A.
Right arrow Articles by Silani, V.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ratti, A.
Right arrow Articles by Silani, V.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Post-transcriptional Regulation of Neuro-oncological Ventral Antigen 1 by the Neuronal RNA-binding Proteins ELAV*Formula

Antonia Ratti{ddagger}1, Claudia Fallini{ddagger}, Claudia Colombrita{ddagger}, Alessia Pascale§, Umberto Laforenza, Alessandro Quattrone||, and Vincenzo Silani{ddagger}

From the {ddagger}Department of Neurological Sciences, "Dino Ferrari" Centre, University of Milan Medical School, IRCCS Istituto Auxologico Italiano, 20095 Cusano, Milan, the Departments of §Experimental and Applied Pharmacology and Experimental Medicine, University of Pavia, 27100 Pavia, and the ||Laboratory of Translational Genomics, Centre for Integrative Biology, University of Trento, 38100 Trento, Italy

Received for publication, July 24, 2007 , and in revised form, January 23, 2008.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Alternative splicing of pre-mRNAs plays an important role in generating biological and functional diversity. Neuro-oncological ventral antigen 1 (Nova1) is a neuron-specific splicing factor that controls the alternative processing of a wide array of mRNAs important for synaptic activity. It is essential for the proper development of the mammalian motor system and for the survival of motoneurons. Because Nova1 gene contains putative regulatory AU-rich elements (ARE) in its highly conserved 3'-untranslated region, we investigated whether its expression is regulated by post-transcriptional mechanisms mediated by ARE-binding proteins. Among these, the neuronal ELAV (nELAV) factors are interesting candidates, because their RNA binding activity is necessary for neuronal differentiation and maintenance. By analysis of ribonucleoprotein complexes in vivo and in vitro we demonstrated that the Nova1 mRNA is a novel target of the nELAV proteins. We defined the nELAV binding site by functional experiments with luciferase reporter gene and Nova1 3'-untranslated region deletion sequences. Gene silencing and overexpression of the nELAV member HuD in motoneuronal NSC34 cells indicate that Nova1 mRNA stability and translation are positively and strongly controlled by the nELAV proteins. In addition, nELAV phosphorylation by a PKC-dependent pathway induces the recruitment of Nova1 mRNA to polysomes. Noteworthy, we found that nELAV proteins are also able to modulate Nova1 splicing activity on its target genes. Our data indicate nELAV proteins as the first factors affecting the expression and activity of the neuronal splicing regulator Nova1 and, consequently, as major candidates for the physiological modulation of Nova1-dependent processing of pre-mRNAs in neurons.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Alternative RNA processing is a general mechanism to generate functionally diverse polypeptides from a single gene, being estimated that at least 74% of the human multiexonic pre-mRNAs undergo alternative splicing (1). Neuro-oncological ventral antigen 1 (Nova1)2 is a neuron-specific RNA-binding protein (RBP), which regulates the alternative splicing of a set of transcripts involved in the formation and activity of the synapses (2, 3). Nova1 null mice exhibit severe defects in the development of the motor system suggesting an essential function of this neuronal splicing factor for the survival and maintenance of motoneurons (4). Nova1 protein is mainly localized in the ventral spinal cord and midbrain, whereas Nova2, the other member of the Nova RBP family, shows a different and complementary expression pattern, being involved in the control of pre-mRNA processing in other two brain regions, the neocortex and the hippocampus (5–7). Nova1 and Nova2 genes are highly homologue and phylogenetically conserved, but profoundly differ in their 3'-untranslated region (3'-UTR) sequences. Whereas the long Nova1 3'-UTR is well conserved in mammals (97% identity between man and mouse) and enriched in regulatory AU-rich consensus elements (ARE), the shorter Nova2 3'-UTR is more variable among orthologs. These observations suggest that post-transcriptional regulation mechanisms may participate in the control of the differential expression of the two Nova genes in the nervous system.

Post-transcriptional regulation mechanisms, indeed, rely on specific cis-acting elements present in the 3'-UTR of target mRNAs, and on RBPs and non-coding RNAs as trans-acting factors that specifically recognize and bind such regulatory elements (8). RBPs are important determinants of gene expression, controlling distinct steps of mRNA life such as splicing, transport, stability, translation, and degradation (9). Together with ubiquitous factors, tissue-specific RBPs are responsible for the development and maintenance of differentiated cells and tissues, strongly influencing their proteome composition (10). RBP-mediated post-transcriptional mechanisms are particularly relevant during embryogenesis and in highly specialized cells such as neurons, because they allow a precise spatio-temporal control of gene expression, a subcellular compartmentalization of mRNAs, and a fast modulation of mRNA translation. The great potentiality and complexity of these regulatory processes are well described by the post-transcriptional operon or RNA regulon model (8, 11). In this model the fate of a single mRNA may be predicted to be differentially and combinatorially controlled by specific and dynamic sets of RBPs in distinct cell conditions and/or developmental stages, as clearly verified in yeast (12).

Among the trans-acting factors potentially involved in the post-transcriptional control of Nova1 gene expression, we identified the highly conserved ELAV (embryonic lethal abnormal vision system) proteins as interesting candidates in the nervous system. In mammals the three ELAV family members, HuB, HuC, and HuD, are selectively expressed in neurons, whereas the fourth member, HuR, is ubiquitous (13). ELAV RBPs act as mRNA stabilizing factors and translational enhancers by binding to AREs in the 3'-UTR of target mRNAs that are usually characterized by high turnover rates (14, 15). Different target transcripts of the three neuron-specific ELAV (nELAV) proteins have been identified in the nervous system, such as GAP43 (16), Mapt (17), NF-M (18), AchE (19), and Msi-1 (20). In different cell models, nELAV proteins have been shown to be necessary and sufficient to induce neuronal differentiation (21). This function was also confirmed in HuD null mice by the demonstration that this RBP plays a critical role in regulating the self-renewal capacity and neuronal differentiation of neural stem cells (22). Importantly, also neuronal plasticity and memory formation processes have been shown to be dependent on nELAV RNA-binding activities in the hippocampus (23–25).

In this paper we investigated whether the neuronal splicing factor Nova1, which is essential for motoneuron survival, is a target of the ARE-binding proteins nELAV. In the motoneuronal cell line NSC34 we show that nELAV proteins bind and post-transcriptionally regulate Nova1 mRNA, increasing its stability and translation and, most importantly, modulating Nova1 activity as a splicing factor.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
NSC34 Cell Culture and Treatments—The motoneuronal cell line NSC34 (a kind gift of N. R. Cashman, University of British Columbia, Vancouver, Canada) was cultured in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum, 1 mM sodium pyruvate, 100 units/ml penicillin, and 100 µg/ml streptomycin. Transient and stable transfections were performed with Lipofectamine Plus (Invitrogen) following the manufacturer's instructions. Puromycin (1 µg/ml) was used for selection and maintenance of stable short hairpin RNA (shRNA) transfectants. Neurite length was measured using QWin Plus 3.3 software (Leica Microsystems) on five independent images for a total of more than 600 cells per condition. NSC34 cells were exposed to 50 µM 5,6-dichlorobenzamidazole riboside (DRB), 100 nM phorbol 12-myristate 13-acetate (PMA) or to the solvent (Me2SO) alone for the indicated time. All reagents were purchased from Sigma.

mRNP Isolation and Immunoprecipitation—Isolation of endogenous mRNPs was conducted as already described (20). Briefly, 5 x 106 NSC34 cells were harvested and resuspended in RNP buffer (100 mM KCl, 5 mM MgCl2, 10 mM HEPES pH 7.4, 0.5% Nonidet P-40, 10 µM dithiothreitol) plus a protease inhibitory mixture (Roche). NSC34 lysates (350 µg) were incubated in NT2 buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM MgCl2, 1 mM dithiothreitol, 20 mM EDTA, 0.05% Nonidet P-40) together with protein G-Sepharose beads (GE Healthcare) pre-coated with 5 µg of the pan-neuronal ELAV (16A11, Invitrogen) or the isotopic IgG (Santa Cruz Biotechnology) antibody. An aliquot (10%) of the reaction mixture was collected as the initial input for sample normalization and total RNA was then extracted by TRIzol reagent (Invitrogen) according to the manufacturer's recommendations. mRNAs were phenol-chloroform extracted from immunoprecipitated mRNPs after digestion with proteinase K for 30 min, retrotranscribed, and quantitated by real time PCR. Data were analyzed as previously reported (20).

Real Time Quantitative PCR—mRNA retro transcription was performed after DNase I (Roche) treatment using SuperScriptII RT (Invitrogen) and oligo(dT) primers. Oligonucleotide pairs for each gene were designed with Primer Express 2.0 software (Applied Biosystems) on exon boundaries (for primer sequences see supplemental materials Table 1). Real time PCR was performed for 40 cycles with SYBR Green PCR Master mixture (Applied Biosystems) and processed on ABI Prism 7900HT (Applied Biosystems). Reactions were run in triplicate for each sample and a dissociation curve was generated at the end. Threshold cycles (Ct) for each tested gene were normalized on the housekeeping Rpl10a gene value ({Delta}Ct) and every experimental sample was referred to its control ({Delta}{Delta}Ct). -Fold change values were expressed as 2–{Delta}{Delta}Ct.

Plasmid Constructs—Nova1 3'-UTR full-length and deletion fragments were RT-PCR amplified from mouse total brain RNA and cloned in pCRII vector (Invitrogen). For luciferase activity assays, Nova1 A1 deletion fragments were XbaI cloned 3' of the Renilla reporter gene in the pGL4.71P vector (Promega). For shRNA gene silencing assay, a double-stranded oligonucleotide complementary and specific for the murine HuD sequence was BglII/HindIII cloned into the pSUPER.retro vector modified in the linker region (a kind gift of G. Pelicci, IEO, Milan, Italy). All primer sequences are reported in supplemental materials Table 2. All clones and their orientation were validated by sequencing. Single nELAV cDNAs and the corresponding recombinant proteins were obtained as previously described (20). The pEGFP-HuD plasmid containing the human HuD cDNA was a kind gift of N. I. Perrone-Bizzozero (University of New Mexico, Albuquerque, NM).

In Vitro Transcription, RNA Electrophoretic Mobility Shift Assays (REMSA), and UV Cross-linking—Radiolabeled riboprobes were obtained by transcribing 0.5 µg of restriction enzyme-linearized construct DNA in the presence of 20 µCi of [{alpha}-32P]UTP as already described (20). The resulting riboprobes were purified on ProbeQuant G-50 microcolumns (GE Healthcare). For REMSA experiments, 300,000 cpm of 32P-labeled riboprobe were incubated with 10 ng of each recombinant nELAV protein for 10 min at room temperature in 25 µl of binding buffer (150 mM NaCl, 20 mM Tris-HCl, pH 7.4, 1.5 mM MgCl2, 0.5 mM dithiothreitol, 5% glycerol, and 0.5 µg/µl tRNA). For competition assays, 2- and 4-fold excess of unlabeled RNA was preincubated for 20 min with a mixture of the three recombinant nELAV proteins prior to the addition of the radioactive Nova1 riboprobe. Samples were then run on a 5% PAGE in TBE buffer and visualized by autoradiography. UV cross-linking and immunoprecipitation experiments were performed as previously described (20) using the pan-neuronal ELAV (Invitrogen) or the irrelevant IgG antibody.

HEK293T Cell Culture and Luciferase Assay—Human embryonic kidney (HEK) 293T cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. For each experimental condition, 300,000 cells were plated in duplicate and transfected using 2 µl of Lipofectamine Plus and 200 ng of each construct (Renilla-A1 subclones and pEGFP-HuD) and 200 ng of firefly reporter vector (pGL3P, Promega). Measurement of the luciferase activity was performed using the Dual Luciferase Reporter Assay System (Promega) 24 h after transfection. The Renilla luciferase activity of the Nova1 3'-UTR constructs was normalized against the firefly luciferase output.

Protein Extraction, Immunoprecipitation, and Western Blotting—Total mouse brain and NSC34 cells were homogenized in lysis buffer (150 mM NaCl, 20 mM Tris-HCl, pH 7.4, 1% Triton X-100, protease inhibitor mixture). Samples were centrifuged at 12,000 x g for 15 min at 4 °C and supernatants were collected. Proteins from different cell fractions were obtained as previously described (26). Proteins were resolved on 10% SDS-PAGE, transferred to nitrocellulose membrane, and immunoblotted with Nova1 (Upstate Biotechnology), PKC{alpha} (Transduction Laboratories), HuR, {alpha}-tubulin, phosphothreonine (Santa Cruz Biotechnology), and phosphoserine (Sigma) antibodies. The capability of the HuR antibody to also detect nELAV proteins was tested on tissues of different origin (supplemental materials Fig. S1). Densitometric analyses were performed with Quantity One software (Bio-Rad).

Analysis of Alternative Splicing Isoforms—Total RNA, isolated from NSC34 stable transfectants using TRIzol reagent, was treated with DNase I and retrotranscribed as described above. The obtained cDNA was quantified by real time PCR using primers for the housekeeping Rpl10a gene so that an equal amount for each sample was employed in the subsequent PCR amplification. Primers for Neogenin and Jnk2 splicing isoforms were designed as previously reported (27, 28). 50 pmol of each forward primer were radiolabeled with 20 µCi of [{gamma}-32P]dATP and 10 units of T4 polynucleotide kinase (Roche). PCR amplification was then performed using 300 nM of each primer and Taq Gold DNA polymerase (Applied Biosystem) for 25–30 cycles. Amplicons were run on 6% PAGE in TBE buffer and dried gels were visualized by autoradiography. For detection of Jnk2 splicing isoforms, PCR products were digested with RsaI restriction enzyme (Roche) prior to PAGE as described (28).

Isolation of Polysomes—Polysomes were isolated from 1 x 107 NSC34 cells by a sucrose discontinuous density gradient (1.3/1.9/2.5 M) as described (29). After ultracentrifugation at 90,000 x g for 5 h, the 1.3/1.9 M interface representing the polysomal fraction was collected. The sample was then diluted in gradient buffer (150 mM KCl, 50 mM Tris-HCl, pH 7.4, 5 mM MgCl2) and ultracentrifuged at 100,000 x g for 8 h. Total RNA was immediately isolated from the final pellet using TRIzol reagent.


Figure 1
View larger version (14K):
[in this window]
[in a new window]

  		FIGURE 1.
Analysis of endogenous nELAV-containing mRNPs in NSC34 cells. The selective immunoprecipitation of nELAV-mRNP complexes was conducted on NSC34 cell lysates using the pan-neuronal ELAV antibody (16A11). The isotypic IgG antibody was used as a control. Co-precipitated transcripts were quantified by real time RT-PCR assay with specific primers for Nova1, Gap43, Mapt, and Rpl10a genes. For each immunoprecipitated sample, normalization was conducted on the mRNA amount of each gene in the initial lysate. -Fold change was calculated versus the control IgG antibody. Values are expressed as mean ± S.E. (n = 4; one-way analysis of variance and Tukey post hoc test; *, p < 0.05; **, p < 0.01).

 
Immunofluorescence—Cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (pH 7.4) for 20 min, blocked with 10% normal goat serum (Invitrogen), and permeabilized with 0.3% Triton X-100. The pan-neuronal ELAV (1:40) and Nova1 (1:500) antibodies were used for 70 min at 37 °C and detected by Cy2- and Cy3-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories). Nuclei were visualized by 4',6-diamidino-2-phenylindole staining. As a negative control, primary antibodies were replaced by normal goat serum. Slides were mounted with Fluorsave (Calbiochem) and acquired by optical (DMIRE2/HCS, Leica Microsystems) and confocal (CS SP2 AOBS, Leica Microsystems) microscopes.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
nELAV Proteins Associate with Nova1 mRNA in Vivo—The murine Nova1 3'-UTR sequence is very long (5.4 kb) and is 97% identical to the shorter 1.9-kb long human ortholog. This highly conserved region contains putative ARE signatures scattered along the entire sequence. Its secondary structure was predicted by the Sfold algorithm (30) to be very stable (–505 kcal/mol) and hairpin-rich (data not shown), suggesting the potential binding of trans-acting regulatory factors.

We used a ribonomic approach described by Tanenbaum et al. (31) to isolate ribonucleoprotein (mRNP) complexes that physiologically form in live cells. To examine whether nELAV proteins and the Nova1 transcript are associated in the motoneuronal NSC34 cells, nELAV-containing mRNP complexes were selectively immunoprecipitated with a pan-neuronal antibody recognizing the three nELAV RBPs HuB, HuC, and HuD. The co-precipitated mRNAs were isolated and the presence of the Nova1 transcript was tested by real time PCR quantitation. Nova1 mRNA was enriched by 5.1-fold in the nELAV-containing mRNP complexes in comparison to the control sample immunoprecipitated by the irrelevant IgG antibody (Fig. 1). We tested the presence of two other well known nELAV targets, Gap43 and Mapt, and we observed the enrichment of their transcripts by 3.9- and 2.9-fold, respectively. These data confirm that nELAV mRNP particles were specifically isolated in the assay. Conversely, the fact that the abundant Rpl10a mRNA, whose 3'-UTR sequence has no evident ARE signature, was not enriched in nELAV-containing mRNPs clearly indicates that Nova1 transcript is specifically associated to nELAV proteins in motoneuronal NSC34 cells.


Figure 2
View larger version (109K):
[in this window]
[in a new window]

  		FIGURE 2.
nELAV proteins specifically bind Nova1 mRNA. A, the three recombinant neuronal proteins HuB, HuC, HuD were used in REMSA with 32P-labeled riboprobe corresponding to the murineNova1 3'-UTR sequence (full-length, 1897 nucleotides) highly homologous to the human one (see also Fig. 3A). Green fluorescent protein (GFP) was used as a negative control (first lane). B and C, competition experiments were performed using a mixture of the three HuB, HuC, and HuD proteins (nELAV). As competitors, increasing amounts (2 and 4 times) of Nova1 unlabeled riboprobe (second and third lanes in B, respectively) and of the irrelevant tRNA probe (second and third lanes in C, respectively) were used. Arrowhead indicates the retarded migration of the Nova1 mRNA-nELAV protein complex; asterisk shows free Nova1 riboprobe.

 
Nova1 3'-UTR Is a Target of the ARE-binding Proteins nELAV—We have shown that nELAV RBPs specifically interact with Nova1 mRNA in vivo. To demonstrate that nELAV binding is dependent on Nova1 3'-UTR sequence, we performed REMSA using recombinant proteins for each nELAV RBP and, as radiolabeled riboprobe, the highly conserved 1.9-kb long fragment of mouse Nova1 3'-UTR. A RNA-protein complex clearly formed with all three nELAV RBPs, but not with the GFP protein used as a negative control (Fig. 2A). nELAV specific binding to Nova1 was further tested in competition experiments, where increasing amounts of unlabeled Nova1 3'-UTR mRNA were able to affect the formation of radiolabeled mRNP complexes (Fig. 2B). The specificity of Nova1 mRNA-nELAV protein interaction was also confirmed when an excess of an unrelated and unlabeled RNA probe was used. In this case no effect on the formation of the Nova1-nELAV mRNP complex was observed in REMSA experiments (Fig. 2C).

Functional Mapping of nELAV Binding to Nova1 3'-UTR—To identify the Nova1 sequence responsible for nELAV protein binding, we performed UV cross-linking and immunoprecipitation assays with different deletion fragments of the Nova1 3'-UTR used in REMSA experiments (Fig. 3A). Two partially overlapping fragments, A and B (about 1.1 kb in length), were used as radiolabeled riboprobes in the presence of mouse brain protein lysate. Gap43 3'-UTR mRNA was employed as a positive control in the assay. After selective immunoprecipitation of nELAV-containing mRNPs and SDS-PAGE of the stably formed radiolabeled complexes, a positive band was visualized with fragment A (Fig. 3B). To better map the Nova1 A binding region, two smaller fragments, A1 and A2, were subsequently used in the assay and only A1 riboprobe was shown to be responsible for nELAV protein binding. The computational analysis of this 435-nucleotide long sequence revealed the presence of 5 scattered putative AREs, of whom only one shows the canonical AUUUA sequence (supplemental materials Fig. S2). We therefore generated three smaller deletion fragments according to the distribution of the predicted AREs and used them in UV cross-linking/immunoprecipitation experiments. Only the 213-bp long deletion fragment A1-(221–435) revealed a positive binding to nELAV proteins (Fig. 3B).

To test whether nELAV binding to this region also had functional relevance, we performed a luciferase assay using the three different Nova1 A1 deletion sequences inserted into the unique XbaI cloning site at the 3' of the Renilla reporter gene. Because the A1-(221–435) fragment contained a XbaI recognition site, we obtained and cloned a smaller 123-bp long fragment (A-(221–345)), in which one of the three predicted AREs was excluded (supplemental materials Fig. S2). We used the human HEK293T cells because they do not express the nELAV proteins, as we could assess by Western blot analysis (data not shown). After transient transfection with the Renilla-A1 constructs, we measured the changes of luciferase activity following expression of the pEGFP-HuD plasmid containing the human HuD cDNA. We found that only the shorter A1-(221–345) 3'-UTR sequence could increase the reporter luciferase activity by 1.7-fold when pEGFP-HuD was transfected into HEK293T cells (Fig. 3C).

We thus identified the 123-nucleotide long sequence of Nova1 3'-UTR as the region responsible for the binding of the trans-acting factors nELAV. This region contains a group V ARE sequence (AUUUA) according to the ARE data base (ARED (32)) followed by a non-canonical AUUUUA sequence with a 7-nucleotide GC-rich stretch in-between. The secondary structure prediction of the A1 fragment showed the formation of a loop corresponding to the canonical ARE contained in the A1-(221–345) sequence, whereas the non-canonical one was folded in a less exposed stem structure (data not shown).

HuD Gene Silencing Affects Nova1 Protein Content—In vivo and in vitro experiments indicate that nELAV proteins are able to specifically bind the ARE-containing Nova1 3'-UTR. To obtain functional data about the role of nELAV proteins in regulating post-transcriptionally Nova1 gene expression, we designed a complementary sequence specific to murine HuD mRNA (shHuD) to silence its expression in NSC34 cells by a shRNA plasmid-based technology. After isolation of stable transfectants, examination of shHuD cell morphology revealed a marked phenotypic change with a prominent loss of spontaneous neurite outgrowth and arborization in comparison to both parental and mock-transfected NSC34 cells (supplemental materials Fig. S3, A–C). We found that the proportion of short neurite-bearing cells was significantly increased in shHuD stable transfectants compared with parental and control cells. On the contrary, the subpopulations presenting longer neuronal processes significantly diminished in shHuD cells (supplemental materials Fig. S3D).


Figure 3
View larger version (40K):
[in this window]
[in a new window]

  		FIGURE 3.
Functional mapping of Nova1 3'-UTR sequence responsible for nELAV protein binding. A, schematic representation of human (Hs) and mouse (Mm) Nova1 3'-UTRs. Different deletion fragments were generated to cover the highly conserved murine Nova1 3'-UTR sequence (FL, full-length). The endogenous XbaI restriction site is indicated. B, SDS-PAGE of nELAV-containing mRNP complexes immunoprecipitated by the pan-neuronal ELAV antibody after UV irradiation in the presence of radiolabeled Gap43 3'-UTR (+, first lane) or Nova1 deletion fragments. The isotypic IgG antibody was used as a negative control (–, last lane) with a mixture of Nova1 A1-{Delta}284, A1-{Delta}251, and A1-(221–435) riboprobes. The arrowhead indicates a nELAV-containing mRNP complex with an apparent molecular mass of 40 kDa. C, luciferase activity of the three different Renilla-A1 constructs in HEK293T cells in the absence (Ctrl) or presence of pEGFP-HuD vector. Normalized Renilla luciferase activity is represented relative to control cells. Values are expressed as mean ± S.E. (n = 6; one-way analysis of variance and Tukey post hoc test; ***, p < 0.001).

 
We evaluated the effective HuD gene silencing in the selected shHuD clone by real time RT-PCR and Western blot analyses. Besides the 55% down-regulation of the HuD mRNA level, we also found that HuB and HuC gene expression was greatly decreased (Fig. 4A). To assess whether such down-regulation was selectively restricted to the nELAV transcripts, we evaluated the expression of the ubiquitous HuR and TIA-1 RBP mRNAs. HuR was only partially affected in shHuD cells, whereas no change in the level of the abundant ELAV-related TIA-1 RBP was observed. In line with the mRNA data, Western blot assay confirmed that the amount of nELAV proteins was also consistently decreased in shHuD in comparison to parental and control cell lysates (Fig. 4B).

We then investigated the effect of HuD gene silencing on Nova1 gene expression by measuring its protein level. Noticeably, we found that the content of Nova1 protein was significantly reduced to 53.5% in shHuD compared with parental NSC34 cells (Fig. 4, C and D). To further confirm the nELAV-dependent post-transcriptional regulation of Nova1 mRNA, HuD gene expression was restored in the knocked-down shHuD cells by transient transfection with the pEGFP-HuD plasmid. We observed that, together with an overall up-regulation of nELAV RBPs, re-expression of the HuD gene also determined the increase of Nova1 protein compared with mock-transfected cells (Fig. 4C). The human HuD cDNA of the pEGFP-HuD construct has a 2-base pair difference with the murine shRNA target sequence. The effective expression of pEGFP-HuD mRNA and the absence of its inhibition by the shRNA construct in shHuD cells were confirmed by RT-PCR using oligonucleotides encompassing the pEGFP vector and the human HuD cDNA sequence (supplementary data Fig. S4). These functional assays show that nELAV RBPs act as strong determinants of Nova1 protein content through post-transcriptional regulation of its mRNA.


Figure 4
View larger version (49K):
[in this window]
[in a new window]

  		FIGURE 4.
Effect of HuD knock-down on Nova1 gene expression. A, real time RT-PCR analysis of Elav and TIA-1 mRNAs in parental (par), mock-transfected (mock), and HuD knocked-down (shHuD) cells. -Fold change values for each gene were calculated versus parental cells (mean ± S.E.; n = 3; one-way analysis of variance and Tukey post hoc test; **, p < 0.01; ***, p < 0.001). B and C, representative Western blots of Nova1 and ELAV proteins in parental, mock, and shHuD cells, and in shHuD clone after transfection with pEGFP-HuD vector (shHuD + HuD). The specificity of the ELAV antibody used in Western blot was first assessed on lysates from different tissues (supplemental materials Fig. S1).{alpha}-Tubulin is used for sample normalization. D, densitometric analyses of Nova1 Western blot data normalized to {alpha}-tubulin (mean ± S.E.; n = 4; one-way analysis of variance and Tukey post hoc test; *, p < 0.05; **, p < 0.01).

 


Figure 5
View larger version (50K):
[in this window]
[in a new window]

  		FIGURE 5.
The splicing activity of Nova1 is altered in shHuD cells. The splicing patterns of (A) Jnk2 (exon 6) and (B) Neogenin (exon 27) genes were analyzed by radiolabeled RT-PCR and autoradiography. A representative gel is shown (left panels) and the relative amount of the splicing isoforms in control (mock), shHuD, and shHuD + HuD cells is visualized in the graph (right panels, mean and S.E.; n = 4). An equal amount of cDNA was used for each amplification reaction after quantitation of the housekeeping Rpl10a transcript in real time PCR experiments.

 
Nova1 Splicing Activity Depends on nELAV Protein Level—After finding that Nova1 protein content is strictly dependent on the upstream levels of the trans-acting factors nELAV, we examined whether such nELAV-mediated post-transcriptional regulation is also able to affect the splicing activity of Nova1 RBP on its target pre-mRNAs. The neuron-specific processing of Jnk2 and Neogenin genes has been previously shown to be controlled by Nova1 in the mouse brain (28). We studied and compared the alternative splicing pattern of these two genes in shHuD and mock-transfected motoneuronal NSC34 cells by performing RT-PCR assays with radiolabeled oligonucleotides. The predominant splicing variant of both tested genes was the unexpected non-neuronal one in mock-transfected (Fig. 5) as well as in parental cells (data not shown). When we determined the Jnk2 gene splicing pattern in shHuD cells, we found that the neuron-specific isoform, containing exon 6a, decreased compared with the control clone (9 versus 22%, respectively). The ratio between the two spliced isoforms changed and returned to be similar to the control clone when the pEGFP-HuD plasmid was transiently transfected to restore HuD gene expression (Fig. 5A). Conversely, when we analyzed the Neogenin splicing pattern, we found that the neuronal isoform was significantly increased in shHuD cells compared with control (62 versus 22%, respectively) (Fig. 5B). Again, HuD gene expression in shHuD cells re-established the original splicing pattern present in the mock-transfected clone. We also observed that HuD gene re-expression determined a consistent increase of the amount of all the splicing variants of both Jnk2 and Neogenin genes. These data show that nELAV proteins are able to influence indirectly the splicing activity of Nova1 RBP on its target pre-mRNAs by controlling Nova1 gene expression and protein content at the post-transcriptional level.

nELAV RBPs Act as Stabilizing Factors on Nova1 mRNA—We studied the degradation rate of the endogenous Nova1 mRNA in NSC34 cells in conditions of transcriptional arrest induced by DRB, a compound able to specifically block RNA polymerase II activity. The content of Nova1 mRNA was monitored in a 8-h time course in parental, control, and shHuD cells. As reported in Fig. 6, Nova1 mRNA degradation kinetics was faster in shHuD cells in comparison to both parental and control cells. Indeed, Nova1 mRNA half-life was 2.95 h in shHuD cells, whereas in parental and mock-transfected cells it was 4.37 and 4.95 h, respectively. When the shHuD cells were transiently transfected with the pEGFP-HuD plasmid, the decay rate of Nova1 transcript decreased showing a half-life of 11.24 h. These results clearly indicate that nELAV RBPs act as stabilizing factors on Nova1 mRNA, as already described for other documented nELAV targets (21).


Figure 6
View larger version (23K):
[in this window]
[in a new window]

  		FIGURE 6.
Nova1 mRNA degradation kinetics depends on nELAV protein levels. Nova1 mRNA content was quantified by real time PCR in parental (par), control (mock), shHuD, and shHuD + HuD cells after 2, 4, 6, and 8 h of transcriptional arrest by DRB treatment. Nova1 mRNA amount at each time point was compared with the initial mRNA level (100%). A nonlinear regression analysis was conducted, which gave a first-order decay constant (k; mean and S.E.; n = 6). Average mRNA half-life (t1/2) was calculated as 0.69/k and reported in the table (**, p < 0.01 versus both parental and mock).

 
PKC-{alpha} Activation Induces nELAV Protein Phosphorylation and Increases Nova1 mRNA Translation—We have previously shown in human neuroblastoma SH-SY5Y cells that protein kinase C (PKC) agonists, such as phorbol esters (PMA) and bryostatin, induce the phosphorylation of nELAV RBPs and the translation of the Gap43 mRNA in an nELAV-dependent way (33).

Treatment of NSC34 cells with PMA for 10 min determined the translocation of the PKC-{alpha} protein from the cytosolic to the membrane and cytoskeletal fractions (Fig. 7A). We evaluated the phosphorylation state of nELAV proteins after PMA treatment by selective immunoprecipitation with phosphothreonine and phosphoserine antibodies. A consistent and specific increase of threonine phosphorylation was observed for nELAV RBPs, because HuR protein was unaffected (Fig. 7B). No change in the amount of phosphoserine residues was detected (data not shown). The total amount of nELAV proteins remained unchanged (Fig. 7C), therefore confirming the specific increase of their phosphorylated threonine residues after PKC-{alpha} activation. These results are in line with our previous data obtained in human neuroblastoma cells (33).

In this short time frame, PMA treatment also determined a significant increase (57%) of Nova1 protein steady-state levels (Fig. 7D). In the attempt to estimate the contribution of post-transcriptional regulation to this increase, transcriptional arrest was induced by a 30-min exposure of NSC34 cells to DRB before PMA treatment. Again, we measured a significant up-regulation (38%) of the amount of Nova1 protein, thereby excluding a major transcriptional contribution to the observed effect. Indeed, PMA treatment was shown to determine a 1.8-fold increase in Nova1 mRNA levels, although with no statistical significance (Fig. 7E). However, because DRB completely abolished this PMA-dependent Nova1 mRNA up-regulation, the increase of Nova1 protein content after PKC stimulation should be attributed mainly to post-transcriptional events. Any significant contribution of increased Nova1 mRNA stability should be excluded at 10 min, given the half-life of 4.37 h for Nova1 mRNA in NSC34 cells (see Fig. 6). So we focused on the hypothesis of increased Nova1 mRNA polysomal loading.

We isolated the polysome fraction from NSC34 cells after PMA treatment in conditions of transcriptional arrest, and changes in Nova1 mRNA engagement in translation were measured by real time RT-PCR. Our results revealed a significant 47% increase of Nova1 mRNA content in the polysome fraction after this short time of PMA treatment (Fig. 7F), whereas the association to polysomes of the abundant glyceraldehyde-3-phosphate dehydrogenase mRNA, which is devoid of regulatory AREs in its 3'-UTR, did not change after PKC activation.

Nova1 RBP Is Localized in Neurites in NSC34 Cells—We have shown that Nova1 gene expression is under the control of post-transcriptional regulatory mechanisms mediated by nELAV RBPs. We investigated the subcellular localization of Nova1 and nELAV proteins in NSC34 cells. According to its function as a splicing factor, immunostaining assays revealed a main distribution of Nova1 in the nuclei and in large Cajal body-like granules. Interestingly, a diffuse and granular localization of Nova1 protein was also observed in the cytoplasm and neurites of NSC34 cells (Figs. 8, A & D) with an overlapping expression pattern with nELAV RBPs (Fig. 8, B and E). However, confocal imaging suggested coexpression, but not colocalization, of Nova1 and nELAV proteins in neurites (Fig. 8, C and F).

To examine the possible interaction between these RBPs, coimmunoprecipitation experiments were carried out. No coprecipitation of these neuronal RBPs was detected in NSC34 cells lysates using both nELAV and Nova1 antibodies (data not shown), confirming the confocal imaging data.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In the mammalian nervous system alternative processing of pre-mRNAs is a very important regulatory mechanism as it allows to modulate protein composition according to different stimuli and conditions (34). In particular, alternative splicing of neuron-specific isoforms actively participates in creating and maintaining the architectural and functional complexity of the brain (35). Nonetheless, few neural factors regulating alternative splicing have been identified and characterized so far. One such neuron-specific splicing factor is Nova1, whose activity has been shown to be necessary for the development of the motor system and the survival of motoneurons (4).


Figure 7
View larger version (43K):
[in this window]
[in a new window]

  		FIGURE 7.
Phorbol ester-induced activation of nELAV proteins promotes Nova1 mRNA translation. A, representative Western blot showing the content of PKC-{alpha} isozyme in NSC34 cell fractions (cytosol, membranes, and cytoskeleton) in parental cells (Ctrl) and after a 10-min treatment with phorbol esters (PMA). {alpha}-Tubulin was used for sample normalization. B, Western blot of nELAV proteins after immunoprecipitation with anti-phosphothreonine antibody in control and PMA-treated cells. Arrowheads indicate the antibody heavy and light chains. C, immunoblot of total nELAV proteins in untreated (Ctrl) and PMA-treated NSC34 cells. D, left panel, representative Western blot of Nova1 protein in control (first and third lanes) and PMA-treated cell lysates (second and fourth lanes) in the presence (third and fourth lanes) or absence (first and second lanes) of the transcription inhibitor DRB. Right panel, densitometric analysis of immunoblot data normalized to {alpha}-tubulin (mean ± S.E.; n = 3; Student's t test; *, p < 0.05; **, p < 0.01). E, real time PCR quantification of Nova1 mRNA in control and PMA-treated cells with or without DRB pre-treatment (mean ± S.E.; n = 3; Student's t test). F, Nova1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNAs, isolated from the polysome-enriched fraction, were quantified by real time PCR in conditions of transcriptional arrest with DRB and after stimulation with PMA (mean ± S.E.; n = 3; one-way analysis of variance and Tukey post hoc test; **, p < 0.01; ***, p < 0.001). The housekeeping Rpl10a mRNA was used for sample normalization.

 
Because Nova1 controls the alternative splicing of a wide array of pre-mRNAs involved in the formation and activity of the synapses, we expect its gene expression to be tightly regulated. Gene expression is first determined by transcription factors, but post-transcriptional mechanisms triggered by RBPs and microRNAs have recently been revealed to be essential to guarantee its fast, precise, and complex regulation in the brain (36). The presence of several putative AREs in the highly conserved 3'-UTR of the mammalian Nova1 genes suggests that post-transcriptional mechanisms mediated by ARE-binding proteins may control Nova1 mRNA stability and translation. We speculate that the differential evolutionary conservation of the Nova1 and Nova2 3'-UTR sequences might well reflect the distinct expression pattern and function of these two genes in the mammalian brain. Among the possible ARE-binding proteins regulating Nova1 mRNA at the post-transcriptional level, the ELAV family is of great interest because three of the four members are specifically expressed in neurons with an overlapping pattern to Nova1 in the spinal cord (4, 37, 38). In this paper we have demonstrated that the neuronal ELAV (nELAV) mRNA-stabilizing RBPs specifically recognize and bind Nova1 3'-UTR in an ARE-containing 123-nucleotide long region.


Figure 8
View larger version (48K):
[in this window]
[in a new window]

  		FIGURE 8.
Localization of Nova1 and nELAV RBPs in motoneuron-like NSC34 cells. Immunofluorescence images of Nova1 (A and D, red) and nELAV (B and E, green) protein localization in NSC34 cells. Merged images show strong coexpression signals in cytoplasm, dendrites, and axons (C and F). G, negative control for immunostaining was obtained by replacing primary antibodies with normal goat serum. Nuclei were counter-stained by 4',6-diamidino-2-phenylindole (blue). Scale bars, 25 µm in A–C and 20 µm in D–G.

 
By performing HuD gene silencing experiments in the motoneuronal NSC34 cells, we observed marked alterations in cell morphology, as already demonstrated in vitro for other neuronal cell models and in HuD null mice (13). These data clearly suggest that nELAV proteins are important for neuronal differentiation and neurite outgrowth also in motoneuronal cells. Interestingly, we observed that the HuD-specific gene silencing also decreased the expression of the other two nELAV members, HuB and HuC, but not of the ubiquitously expressed HuR and TIA-1 mRNAs. This suggests the existence of autoregulatory mechanisms that, in a crossed manner, may control the expression of all neuronal members of this RBP family in the brain. This autoregulatory mechanism has already been described for HuB and for the elav gene in Drosophila, whose ARE-containing transcripts are controlled by their own gene products (39–41). Interestingly, it has been recently demonstrated that several ubiquitous RBPs, including HuR, reciprocally influence and regulate post-transcriptionally their expression in a complex crossed-autoregulatory network (42).

The important finding is that HuD gene down-regulation caused a consistent decrease of Nova1 protein level. The demonstration that this effect on Nova1 was due to the activity of nELAV proteins came from the observation that re-expression of the HuD gene in shHuD cells was able to restore Nova1 protein content. The molecular explanation of this effect is that nELAV RBPs act as stabilizing factors on Nova1 mRNA, whose turnover rate we showed to be inversely correlated to the expression level of nELAV proteins.

Our data also provide suggestive evidence that nELAV proteins favor the translation of the bound Nova1 target transcript. A direct association of nELAV RBPs to ribosomes and the translating machinery has been previously documented (18, 25). Furthermore, activation of nELAV proteins by a PKC-dependent pathway determines their translocation to the cytoskeletal compartment (20, 33), which has been shown to be important for the interaction with the translational apparatus (43). In NSC34 cells we observed that the acute (10 min) PKC-mediated phosphorylation of nELAV proteins was associated to a specific recruitment of Nova1 mRNA to the polysomal fraction. We can be confident that this is a mere post-transcriptional regulation of the Nova1 transcript because we prevented the effects of PMA, a classical PKC activator, on the transcriptional activity by using a specific chemical inhibitor of RNA polymerase II. Therefore the observed early increase of Nova1 protein content is basically dependent on the increased availability of its mRNA to ribosomes, although some indirect PMA effects on the stability of Nova1 protein itself cannot be completely excluded. Taken together, our results are strongly in agreement with a direct positive effect of activated nELAV proteins on Nova1 mRNA translatability.

It is noteworthy to observe that the nELAV-dependent regulation of Nova1 gene expression is also able to influence its function as a splicing factor. In fact, the splicing patterns of two Nova1 target pre-mRNAs were found to be altered following Nova1 down-regulation in shHuD cells. We observed an opposite effect on the splicing of the two neuronal-specific isoforms of Jnk2 and Neogenin genes. This phenomenon may in part be explained by the experimental cell model we used, which is embryonic in origin, whereas previous data on Nova1 splicing activity were obtained from the postnatal neocortex of Nova null mice (28). Moreover, we speculate that the decrease of Nova1 protein has distinct or opposite effects on different transcripts depending on the specific RBPs required for their processing. Indeed, the neuron-specific splicing of pre-mRNAs is regulated in various neuronal cell populations by different RBPs that have complementary, additive or even competitive functions, as already demonstrated for brPTB and Nova1 (44).

The fact that HuD gene re-expression in shHuD cells was able to completely restore the splicing patterns of Jnk2 and Neogenin genes may be directly associated to the nELAV-mediated up-regulation of Nova1 protein content by increased Nova1 mRNA stability and translatability. Interestingly, in condition of HuD overexpression, we also observed an overall increase of the Jnk2- and Neogenin-spliced isoforms. This finding seems to correlate with the presence of putative AREs in the 3'-UTR of these genes,3 indicating a possible nELAV-dependent stabilizing effect also on Nova1 target mRNAs.

Our data on Nova1 protein localization in NSC34 neurites suggest the fascinating hypothesis that Nova1 RBP may have an additional role to its well documented splicing activity, and may also be involved in the transport or translational control of target mRNAs. Although further evidence needs to be gained from in vivo studies, this possibility is not so remote. It is known from the literature that a single RBP may have multiple functions related to the mRNA metabolism, as in the case of the nELAV proteins themselves, which are known to be involved in mRNA stabilization and translation as well as in splicing and transport activities (17, 45, 46). In particular, in motoneurons the SMN mRNP complex, responsible for the assembly and function of the splicing machinery, is also involved in the axonal transport of mRNAs (47).

All our results confirm that post-transcriptional mechanisms mediated by nELAV proteins exert a pivotal neurobiological role in the control not only of Nova1 mRNA stability and translation but, importantly, also of its splicing function. We have shown that a change in nELAV protein abundance may dramatically affect the correct processing of Nova1 target pre-mRNAs, with potential long-term consequences on the overall metabolism and activity of neurons. Our data are the first ones identifying post-transcriptional factors that can effectively modulate Nova1 RBP abundance and splicing activity in the nervous system, although we suppose that such regulation is more complex and likely requires the competitive and/or synergic action of other RBPs and microRNAs.

In conclusion, here we have functionally correlated two important classes of neuron-specific RBPs, nELAV and Nova, which interestingly represent the autoantigens of two distinct paraneoplastic neurologic syndromes, encephalomyelitis and paraneoplastic opsoclonus myoclonus ataxia, respectively (48, 6). The identification of nELAV proteins as pivotal post-transcriptional regulators of Nova1 gene expression and activity may help elucidate the molecular mechanisms underlying the neurobiology of highly specialized cells such as motoneurons. The function of nELAV proteins in motoneurons has never been investigated so far, even if their expression has been observed in the ventral spinal cord in vertebrates (37, 49). These findings also suggest that alterations in the nELAV-mediated post-transcriptional regulatory mechanisms may severely affect the survival of motoneurons and, eventually, contribute to the development of motoneuron diseases in humans.


   FOOTNOTES
 
* This work was supported by Italian Ministry of Health Malattie Neurodegenerative, ex Art.56, Grant 533F/N 1, 2004. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables 1 and 2 and Figs. S1–S4. Back

1 To whom correspondence should be addressed: IRCCS Istituto Auxologico Italiano, Via Zucchi, 18, 20095 Cusano, Milan, Italy. Tel.: 39-02-619113045; Fax: 39-02-619113033; E-mail: antonia.ratti{at}unimi.it.

2 The abbreviations used are: Nova1, neuro-oncological ventral antigen 1; ARE, AU-rich element; 3'-UTR, 3'-untranslated region; ELAV, embryonic lethal abnormal vision system; RBP, RNA-binding proteins; REMSA, RNA electrophoretic mobility shift assays; shRNA, short hairpin RNA; DRB, 5,6-dichlorobenzamidazole riboside; PMA, phorbol 12-myristate 13-acetate; RT, reverse transcriptase; HEK, human embryonic kidney; PKC, protein kinase C. Back

3 A. Ratti, C. Fallini, C. Colombrita, A. Pascale, U. Laforenza, A. Quattrone, and V. Silani, unpublished observations. Back


   ACKNOWLEDGMENTS
 
We thank N. R. Cashman (University of British Columbia, Vancouver, Canada) and A. Poletti (University of Milan, Milan, Italy) for NSC34 cells; N. I. Perrone-Bizzozero (University of New Mexico, Albuquerque, NM) for providing the pEGFP-HuD plasmid. A particular acknowledgement to C. Bodio for technical assistance and G. Pelicci and B. Ortensi (IEO, Milan) for their kind help in gene silencing experiments. We also thank S. Rodighiero and the CIMAINA Facility, University of Milan, for confocal imaging.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Johnson, J. M., Castle, J., Garrett-Engele, P., Kan, Z., Loerch, P. M., Armour, C. D., Santos, R., Schadt, E. E., Stoughton, R., and Shoemaker, D. D. (2003) Science 302, 2141–2144[Abstract/Free Full Text]
  2. Ule, J., and Darnell, R. B. (2006) Curr. Opin. Neurobiol. 16, 102–110[CrossRef][Medline] [Order article via Infotrieve]
  3. Ule, J., Ule, A., Spencer, J., Williams, A., Hu, J. S., Cline, M., Wang, H., Clark, T., Fraser, C., Ruggiu, M., Zeeberg, B. R., Kane, D., Weinstein, J. N., Blume, J., and Darnell, R. B. (2005) Nat. Genet. 37, 844–852[CrossRef][Medline] [Order article via Infotrieve]
  4. Jensen, K. B., Dredge, B. K., Stefani, G., Zhong, R., Buckanovich, R. J., Okano, H. J., Yang, Y. Y., and Darnell, R. B. (2000) Neuron 25, 359–371[CrossRef][Medline] [Order article via Infotrieve]
  5. Yang, Y. Y., Yin, G. L., and Darnell, R. B. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 13254–13259[Abstract/Free Full Text]
  6. Buckanovich, R. J., Yang, Y. Y., and Darnell, R. B. (1996) J. Neurosci. 16, 1114–1122[Abstract/Free Full Text]
  7. Graus, F., Rowe, G., Fueyo, J., Darnell, R. B., and Dalmau, J. (1993) Neurosci. Lett. 150, 212–214[CrossRef][Medline] [Order article via Infotrieve]
  8. Keene, J. D. (2007) Nat. Rev. Genet. 8, 533–543[CrossRef][Medline] [Order article via Infotrieve]
  9. Moore, M. J. (2005) Science 309, 1514–1518[Abstract/Free Full Text]
  10. Perrone-Bizzozero, N., and Bolognani, F. (2002) J. Neurosci. Res. 68, 121–126[CrossRef][Medline] [Order article via Infotrieve]
  11. Keene, J. D., and Tenenbaum, S. A. (2002) Mol. Cell 9, 1161–1167[CrossRef][Medline] [Order article via Infotrieve]
  12. Gerber, A. P., Herschlag, D., and Brown, P. O. (2004) PLoS Biol. 2, E79[CrossRef][Medline] [Order article via Infotrieve]
  13. Pascale, A., Amadio, M., and Quattrone, A. (2008) Cell Mol. Life Sci. 65, 128–140[CrossRef][Medline] [Order article via Infotrieve]
  14. Antic, D., and Keene, J. D. (1997) Am. J. Hum. Genet. 61, 273–278[Medline] [Order article via Infotrieve]
  15. Barreau, C., Paillard, L., and Osborne, H. B. (2005) Nucleic Acids Res. 33, 7138–7150[CrossRef][Medline] [Order article via Infotrieve]
  16. Chung, S., Eckrich, M., Perrone-Bizzozero, N., Kohn, D. T., and Furneaux, H. (1997) J. Biol. Chem. 272, 6593–6598[Abstract/Free Full Text]
  17. Aranda-Abreu, G. E., Behar, L., Chung, S., Furneaux, H., and Ginzburg, I. (1999) J. Neurosci. 19, 6907–6917[Abstract/Free Full Text]
  18. Antic, D., Lu, N., and Keene, J. D. (1999) Genes Dev. 13, 449–461[Abstract/Free Full Text]
  19. Deschenes-Furry, J., Angus, L. M., Belanger, G., Mwanjewe, J., and Jasmin, B. J. (2005) Chem. Biol. Interact. 157–158, 43–49[CrossRef][Medline] [Order article via Infotrieve]
  20. Ratti, A., Fallini, C., Cova, L., Fantozzi, R., Calzarossa, C., Zennaro, E., Pascale, A., Quattrone, A., and Silani, V. (2006) J. Cell Sci. 119, 1442–1452[Abstract/Free Full Text]
  21. Deschenes-Furry, J., Perrone-Bizzozero, N., and Jasmin, B. J. (2006) Bioessays 28, 822–833[CrossRef][Medline] [Order article via Infotrieve]
  22. Akamatsu, W., Fujihara, H., Mitsuhashi, T., Yano, M., Shibata, S., Hayakawa, Y., Okano, H. J., Sakakibara, S., Takano, H., Takano, T., Takahashi, T., Noda, T., and Okano, H. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 4625–4630[Abstract/Free Full Text]
  23. Quattrone, A., Pascale, A., Nogues, X., Zhao, W., Gusev, P., Pacini, A., and Alkon, D. L. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 11668–11673[Abstract/Free Full Text]
  24. Pascale, A., Gusev, P. A., Amadio, M., Dottorini, T., Govoni, S., Alkon, D. L., and Quattrone, A. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 1217–1222[Abstract/Free Full Text]
  25. Bolognani, F., Merhege, M. A., Twiss, J., and Perrone-Bizzozero, N. I. (2004) Neurosci. Lett. 371, 152–157[CrossRef][Medline] [Order article via Infotrieve]
  26. Pascale, A., Fortino, I., Govoni, S., Trabucchi, M., Wetsel, W. C., and Battaini, F. (1996) J. Neurochem. 67, 2471–2477[Medline] [Order article via Infotrieve]
  27. Dredge, B. K., Stefani, G., Engelhard, C. C., and Darnell, R. B. (2005) EMBO J. 24, 1608–1620[CrossRef][Medline] [Order article via Infotrieve]
  28. Ule, J., Jensen, K. B., Ruggiu, M., Mele, A., Ule, A., and Darnell, R. B. (2003) Science 302, 1212–1215[Abstract/Free Full Text]
  29. Mechler, B. M. (1987) Methods Enzymol. 152, 241–248[Medline] [Order article via Infotrieve]
  30. Ding, Y., Chan, C. Y., and Lawrence, C. E. (2004) Nucleic Acids Res. 32, W135–141[Abstract/Free Full Text]
  31. Tenenbaum, S. A., Lager, P. J., Carson, C. C., and Keene, J. D. (2002) Methods 26, 191–198[CrossRef][Medline] [Order article via Infotrieve]
  32. Bakheet, T., Williams, B. R., and Khabar, K. S. (2006) Nucleic Acids Res. 34, D111–D114[Abstract/Free Full Text]
  33. Pascale, A., Amadio, M., Scapagnini, G., Lanni, C., Racchi, M., Provenzani, A., Govoni, S., Alkon, D. L., and Quattrone, A. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 12065–12070[Abstract/Free Full Text]
  34. Black, D. L., and Grabowski, P. J. (2003) Prog. Mol. Subcell. Biol. 31, 187–216[Medline] [Order article via Infotrieve]
  35. Lipscombe, D. (2005) Curr. Opin. Neurobiol. 15, 358–363[CrossRef][Medline] [Order article via Infotrieve]
  36. Kosik, K. S., and Krichevsky, A. M. (2005) Neuron 47, 779–782[CrossRef][Medline] [Order article via Infotrieve]
  37. Okano, H. J., and Darnell, R. B. (1997) J. Neurosci. 17, 3024–3037[Abstract/Free Full Text]
  38. Wakamatsu, Y., and Weston, J. A. (1997) Development 124, 3449–3460[Abstract]
  39. Abe, R., Yamamoto, K., and Sakamoto, H. (1996) Nucleic Acids Res. 24, 2011–2016[Abstract/Free Full Text]
  40. Samson, M. L. (1998) Genetics 150, 723–733[Abstract/Free Full Text]
  41. Borgeson, C. D., and Samson, M. L. (2005) Nucleic Acids Res. 33, 6372–6383[Abstract/Free Full Text]
  42. Pullmann, R., Jr., Kim, H. H., Abdelmohsen, K., Lal, A., Martindale, J. L., Yang, X., and Gorospe, M. (2007) Mol. Cell. Biol. 27, 6265–6278[Abstract/Free Full Text]
  43. Antic, D., and Keene, J. D. (1998) J. Cell Sci. 111, 183–197[Abstract]
  44. Polydorides, A. D., Okano, H. J., Yang, Y. Y., Stefani, G., and Darnell, R. B. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 6350–6355[Abstract/Free Full Text]
  45. Aronov, S., Aranda, G., Behar, L., and Ginzburg, I. (2002) J. Cell Sci. 115, 3817–3827[Abstract/Free Full Text]
  46. Zhu, H., Hasman, R. A., Barron, V. A., Luo, G., and Lou, H. (2006) Mol. Biol. Cell 17, 5105–5114[Abstract/Free Full Text]
  47. Rossoll, W., Jablonka, S., Andreassi, C., Kroning, A. K., Karle, K., Monani, U. R., and Sendtner, M. (2003) J. Cell Biol. 163, 801–812[Abstract/Free Full Text]
  48. Szabo, A., Dalmau, J., Manley, G., Rosenfeld, M., Wong, E., Henson, J., Posner, J. B., and Furneaux, H. M. (1991) Cell 67, 325–333[CrossRef][Medline] [Order article via Infotrieve]
  49. Park, H. C., Hong, S. K., Kim, H. S., Kim, S. H., Yoon, E. J., Kim, C. H., Miki, N., and Huh, T. L. (2000) Neurosci. Lett. 279, 81–84[CrossRef][Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?



This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
283/12/7531    most recent
M706082200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ratti, A.
Right arrow Articles by Silani, V.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ratti, A.
Right arrow Articles by Silani, V.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2008 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement