Julien Grange

Agnès Belly

Stéphane Dupas

Alain Trembleau

Rémy Sadoul

Yves Goldberg
email: <yves.goldberg@ujf-grenoble.fr

St Phane Dupas

Specific interaction between Sam68 and neuronal mRNAs: implication for the activity-dependent biosynthesis of elongation factor eEF1A

Keywords: Adaptor Proteins, Signal Transducing, genetics, metabolism, Animals, Cells, Cultured, DNA-Binding Proteins, genetics, metabolism, Embryo, Mammalian, Gene Expression Regulation, physiology, Hippocampus, cytology, Humans, Immunoprecipitation, methods, Neurons, metabolism, Peptide Elongation Factor 1, biosynthesis, Protein Binding, physiology, Protein Biosynthesis, RNA, Messenger, metabolism, RNA-Binding Proteins, genetics, metabolism, Rats, Receptors, N-Methyl-D-Aspartate, metabolism, Transfection, methods

Introduction

to these somatodendritic sites (

). These results suggest that Sam68 may play a part in the regulation of those mRNAs Ben [START_REF] Fredj | Depolarization-induced translocation of the RNA-binding protein Sam68 to the dendrites of hippocampal neurons[END_REF] which become translationally activated in response to electrical activity. To test this hypothesis, we have investigated the identity of mRNA species that were bound to Sam68 in cortical neurons in vivo. We found that one major target was Eef1a1, an mRNA known to be activated during long-term synaptic change in the hippocampus. In cultured neurons, depolarisation enhanced the association of both Sam68 and the Eef1a1 mRNA with polyribosomes, and increased the amount of its product, the eEF1A protein. Expression of a dominant-negative Sam68 mutant confined to the cytoplasm was able to lower the amount of eEF1A, supporting the proposal that Sam68 promotes the translation of neuronal Eef1a1.

Materials and Methods

Immunoprecipitation of Sam68-associated RNA and preparation of amplified RNA probes Cortical Sam68-RNA complexes were prepared as previously described (

) Briefly, the cortex from one adult rat [START_REF] Grange | Somatodendritic localization and mRNA association of the splicing regulatory protein Sam68 in the hippocampus and cortex[END_REF] was dissected and lysed at 4 C in polysome dissociation buffer 10 mM Tris-HCl, pH 7.5, 50 mM KCl, 1 mM sodium orthovanadate, 2 °[ mM dithiothreitol (DTT), 100 nM microcystine-LR (Sigma), protease inhibitors (Complete; Roche Molecular) containing 0.5 Triton ] %

X-100, 160 mM sucrose, 30 mM EDTA, pH 8.0 and 400 U/ml RNAsin (Promega, Inc., Madison, WI). After clarification by centrifugation at 82,000 for 20 min at 4 C, the lysate was precleared with protein G Sepharose beads (GE Healthcare), then immunoprecipitated with g °affinity-purified anti-Sam68 IgG (AD-1), or with preimmune protein A-purified IgG from the same rabbit. Immune complexes were collected onto protein G Sepharose beads, washed, and processed for RNA purification using the RNEasy kit (Qiagen France, -" "

Courtaboeuf). The immunoprecipitated polyA RNAs were linearly amplified by cDNA synthesis and T7 polymerase transcription ( + ), using the MessageAmp aRNA Kit (Ambion). According to whether the template RNA originated in the specific [START_REF] Gelder | Amplified RNA Synthesized from Limited Quantities of Heterogeneous cDNA[END_REF] ™ immunoprecipitate or the preimmune control, either Cyanine 3 (Cy3) or Cyanine 5 (Cy5)-labeled UTP was incorporated during

™ ™
transcript synthesis, as per manufacturer s instructions. Transcripts amplified from the specific immunoprecipitate and the corresponding ' control were mixed in equal amounts and used to probe the 15000 cDNAs arrayed on the NIA micro-array ( ). To [START_REF] Tanaka | Genome-wide expression profiling of mid-gestation placenta and embryo using a 15,000 mouse developmental cDNA microarray[END_REF] control for possible biasing by differential incorporation of Cy3 and Cy5, the experiment was repeated with swapping the fluorophores between specific and background transcripts.

"

" " "

Quantitation of hybridization

After hybridization and washing, digital images of the micro-array at 635 nm and 532 nm were acquired by laser scanning confocal microscopy. Fluorescent spots were delineated and quantitated with GenePix software (Axon Instruments). Raw fluorescence values (F) in each channel were converted into their Log . After Cy3 labelling of background RNAs, immunoprecipitation of any given mRNA was 2 considered specific when the value in the Cy5 channel exceeded that in the Cy3 channel by at least 4 Log units (a 16-fold difference).

2

When background RNAs were labelled with Cy5, the cut-off was brought down to 0.5 Log unit (an 1.4-fold difference). This differential 2 cut-off was applied to compensate for the systematic bias in labelling efficiency in favour of Cy5. Note that the requirement for higher Cy3 labelling of specifically immunoprecipitated RNAs is conservative and likely excludes a number of Sam68-associated mRNAs. Z scores were calculated as (Log F <Log F>)/ , where <Log F> is the mean and is the standard deviation, both calculated over the 2 -

2 σ Log2F 2 σ Log2F
entire population of fluorescence values in the relevant channel ( ). Reproducibility of the overall population of z scores [START_REF] Mayne | Gene expression profile of herpesvirus-infected T cells obtained using immunomicroarrays: induction of proinflammatory mechanisms[END_REF] was measured by simple linear regression between experiments. 95 confidence intervals were calculated for the z scores of % Table Ia using t statistics (n 3), and individual mRNAs with lower confidence limit z < -1.0 were rejected for inclusion in the Table; more than = min 80 of these scores had lower limit above zero. Frequencies of GO descriptors were computed from ~4000 functionally described genes % of the microarray (available through

). http://lgsun.grc.nia.nih.gov/cDNA/NIA-CloneSet-GoTermFinder.html

Polymerase chain reactions

Immunoprecipitated or total RNA was reverse-transcribed with the universal primer GGAATTC(T) , and the resulting cDNA was DNA accumulation curve (typically between 25 and 32), and standardized by relating to a scale generated with known amounts of total cDNA. The specificity of the amplification product was confirmed by checking its melting curve with the Light Cycler, and its size on an agarose gel.

Immunoprecipitation of RNA-mycSam68 complex from HEK cells

HEK-293 cells were grown in Dulbecco s modified Minimum Essential Medium, containing 10 fetal calf serum (Invitrogen). The ' % myc-Sam68 expressing plasmid was a kind gift from I. Barlat and B. Tocqu (at the time at Aventis Pharma, Vitry, France). One 6 cm dish é of cells was transfected with either myc-Sam68 or with control vector (containing a CMV promoter). 48 h later, cells were lysed in polysome dissociation buffer and, after clarifying by centrifugation, the lysate was immunoprecipitated with 2 g anti-myc monoclonal μ antibody (12CA5, Santa Cruz) and protein G-Sepharose beads, as described for brain lysates. Total RNA was isolated and amplified by RT-PCR as decribed above.

Neuronal cultures and transfections

Primary cultures of rat hippocampal neurons were prepared from fetal (E19) hippocampi and seeded at a density of 6000 cells/cm 2 onto poly-L-lysine-coated glass coverslips (Marienfeld, Germany), adhering to the procedure described by Goslin and Banker (Banker et ), except that the culture medium was Neurobasal with B27 supplement (Invitrogen). (expressing monomeric red fluorescent protein) was a kind gift from R.Y. Tsien (UCSD, San Diego, CA, USA). Transfection was performed at 9 DIV by calcium phosphate coprecipitation, exactly according to ( ). We used a mix of 2.5 g GFP-Sam68 [START_REF] Goetze | Chemically controlled formation of a DNA/calcium phosphate coprecipitate: application for transfection of mature hippocampal neurons[END_REF] μ and 0.5 g mRFP1 plasmids per coverslip. Expression of the GFPSam68 and GFP-Sam68 C plasmid was stable for the next 10 days.

μ Δ

Neurons were fixed at 14 19 DIV and processed for immunofluorescent staining.

-Primary cortical neurons were prepared from E16 fetal cortices, using essentially the same procedure as for hippocampal neurons. The cortical cells were seeded at a density of 10 cells/cm onto poly-L-lysine coated 10-cm dishes (Nunc). Neurons at 9 DIV were treated for 3 5 2 h with either depolarizing medium (containing,in mM: KCl,25;CaCl2,1.8;MgCl2,0.8;NaCl,100;NaHCO3,26;NaH2PO4,1;glucose,30;15) or control solution (same except for KCl, 5; NaCl, 120).

Sedimentation of polysomes on sucrose gradients

RNA-protein complexes were fractionated as described by ( ). Briefly, cortical neurons were lysed in low [START_REF] Krichevsky | Neuronal RNA granules: a link between RNA localization and stimulation-dependent translation[END_REF] salt buffer 20 mM Tris-HCl (pH 7.5), 10 mM NaCl, 3 mM MgCl2, 1 mM RNasin, 1 mM dithiothreitol, 0.3 Triton X-100, and 0.05 M ml) were overlaid onto 10.6 ml gradients and centrifuged at 32,000 rpm for 2.5 hr at 4 C in a SW41 rotor. Fractions of 1 ml volume were °collected with on-line UV absorbance measurement at 254 nm (GE Healthcare Akta Prime). For Western immunoblotting, the protein content of the fractions was concentrated by trichloroacetic acid precipitation, redissolved in Laemmli buffer, and analysed on 8% polyacrylamide/SDS gels. For RT-PCR, RNA was extracted by treating the relevant fractions with Trizol reagent (Invitrogen), precipitated with ethanol, and subjected to RT-PCR with the same primers as above.

Detection of eEF1A protein increase by immunoblotting

Cortical neurons were treated with control or depolarising medium as above, then lysed in icecold RIPA buffer (20 mM Tris-HCl pH [ 7.5 , 150 mM NaCl, 1 mM EDTA, 1 Triton X-100, 0.5 Na deoxycholate, 0.1 SDS) containing protease inhibitors (EDTA-free

] % % %
Complete, Roche Molecular). After clarification by centrifugation (15 min at 12,000 g), equal protein amounts of each lysate were × analysed by 10 polyacrylamide/SDS gel electrophoresis and immunoblotting, first with anti-eEF1A monoclonal antibody (UBI) then % with anti-actin polyclonal antibody (Santa Cruz). Immunoreactive bands were revealed by chemiluminescence (Pierce Femto) and autoluminography. The films were digitized by scanning and the integrated intensity of the bands was determined using Metamorph software (Universal Imaging). Immunoblotting and exposure conditions were calibrated so that the integrated intensity of the bands was in the linear range with respect to protein amount.

Immunofluorescence of eEF1A in hippocampal neurons

Neurons were fixed with 4 PFA containing 4 sucrose (30 min, 4 C); permeabilized with 0.2 Triton X-100; blocked with 10

% % °% %
preimmune goat serum in PBS (25 C, 1 h); and stained with 1 g/ml anti-eEF1A monoclonal antibody (UBI) in PBS containing 3 goat °μ % serum (25 C, 2 h), then with Alexa350 -conjugated secondary antibody (Molecular Probes). After washing, coverslips were mounted

° ™
with Mowiol and observed on a Zeiss microscope fitted with an epifluorescence setup and a cooled CCD camera (Zeiss Axiocam), or with a confocal laser scanning microscope (Zeiss LSM 510).

For quantitation of eEF1A fluorescence, fields containing transfected neurons were imaged at low magnification (20x). Imaging conditions were identical for all fields. Images were processed with Metamorph (Universal Imaging). For each image, a background image was produced with a low-pass filter and subtracted so as to remove low-frequency fluorescence noise due e.g. to local unevenness in illumination. Next, a lower fluorescence threshold was set, which allowed automatic delineation and analysis of the fluorescent objects (cells), using Integrated Morphometric Analysis. The same threshold was used throughout the entire analysis. The relative eEF1A fluorescence of each transfected neuron was calculated according to the equation: i where eEF1A

is the mean grey level per pixel within the transfected neuron, and <eEF1A> is the average (over 15 transf.,i nontransf surrounding neurons) of the mean grey level per pixel within non-transfected neurons. These IF values were averaged over the entire transf.,i set of transfected neurons, to yield the values of . Fig. 4E Immunoprecipitation of Sam68-NMDA receptor complexes Rat cortical synaptic membranes were prepared by differential centrifugation as described by , and resuspended in [START_REF] Grange | Somatodendritic localization and mRNA association of the splicing regulatory protein Sam68 in the hippocampus and cortex[END_REF] ice-cold buffer containing 4mM Hepes (pH 7.4), 1mM preboiled sodium orthovanadate, 1mM dithiothreitol, 100 nM microcystin (Sigma), and a mix of protease inhibitors ( Complete , Roche). An aliquot of suspension (530 l, 4.5 g/ l total protein) was solubilized by adding " " μ μ μ

1:9 vol. of detergent solution (10 sodium deoxycholate in 0.5M Tris, pH 9.0) and gently agitating for 20 min. Triton X-100 (0.1 final % % concn.) was then added, and the extract was dialyzed overnight against immunoprecipitation buffer (10 mM Tris-HCl, pH 7.4, 100 mM NaCl, 0.1 mM sodium orthovanadate, 5 mM EDTA, 1 mM dithiothreitol, 0.1 Triton X-100, 1 nM microcystin, and protease inhibitors).

%

The dialyzed extract was centrifuged for 30 min at 10 g in a TL-100 tabletop ultracentrifuge (Beckman), and the supernatant (300 g 5 μ protein diluted to 500 l buffer per IP) was used for immunoprecipitation for 3 h with 10 g affinity-purified anti-Sam68 polyclonal μ μ antibody, or preimmune IgG purified from the same rabbit, or with anti-NR1 monoclonal antibody (Pharmingen). Immunoprecipitates were collected onto protein G-Sepharose beads and analysed by SDS-PAGE (8 polyacrylamide gel) and Western immunoblotting.

%

Antibodies used for immunoblotting were purchased from UBI (anti-PSD95 monoclonal antibody) and Chemicon (anti-GluR2/3 rabbit polyclonal antibody). The bands were revealed by chemiluminescence (Pierce Femto).

Results

Identification of in the repertoire of Sam68-associated mRNAs Eef1a1

Following an approach initiated by , we decided to use a combination of co-immunoprecipitation and array [START_REF] Tenenbaum | Identifying mRNA subsets in messenger ribonucleoprotein complexes by using cDNA arrays[END_REF] screening to probe the repertoire of Sam68-associated mRNAs in cortical neurons. Sam68-mRNA complexes were immunoprecipitated from a rat cortical cytoplasmic extract with an anti-Sam68 antibody, as previously reported (

). A parallel Ben [START_REF] Fredj | Depolarization-induced translocation of the RNA-binding protein Sam68 to the dendrites of hippocampal neurons[END_REF] immunoprecipitation was performed with preimmune antibody. The polyA RNAs associated with the Sam68 and control + immunoprecipitates were linearly amplified by cDNA synthesis and T7 polymerase-mediated transcription, as initially described by Eberwine and coworkers ( ), and the amplified transcripts were fluorescently labeled with Cy3 or Cy5, respectively. The [START_REF] Gelder | Amplified RNA Synthesized from Limited Quantities of Heterogeneous cDNA[END_REF] two transcript populations were then mixed and hybridized with the ~15,000 cDNAs of the NIA 15K clone set arrayed onto a glass slide ( " "

), and for each cDNA the fluorescence values at both wavelengths were measured and compared ( ). A given [START_REF] Tanaka | Genome-wide expression profiling of mid-gestation placenta and embryo using a 15,000 mouse developmental cDNA microarray[END_REF] Fig. 1A,B mRNA was considered to be specifically immunoprecipitated when the fluorescence level was consistently higher in the channel corresponding to the immunospecific than in the background precipitation. To compare mRNA abundance measurements across several experiments, normalized measures of the relative abundance of each mRNA in the immunoprecipitated population were calculated by converting the fluorescence values into z scores ( ; ; see Materials and Methods). Duplicate Lopez de [START_REF] Lopez De Silanes | Identification of a target RNA motif for RNA-binding protein HuR[END_REF][START_REF] Mayne | Gene expression profile of herpesvirus-infected T cells obtained using immunomicroarrays: induction of proinflammatory mechanisms[END_REF] assays of the same RNA population showed that z scores were highly reproducible through the entire set of ~15,000 genes, except for a limited number of low abundance mRNAs, indicating that abundance measurements were not grossly perturbed by fluctuations in the hybridization and detection procedure (

). When distinct experiments with different brain samples were compared, fluctuations in Fig. 1C individual z scores were larger, likely due to random differences in the hydrolysis or dissociation rates of individual mRNAs during immunoprecipitation. The correlation between samples remained sufficient to ensure that the species scoring highest (z > 1.5) in one trial retained a 85 probability of scoring positive (z > 0) in a second trial ( ). To obtain reliable identification of the most abundant % Fig. 1D mRNAs in the immunoprecipitate, z scores were measured in three independent experiments, mRNAs were ranked by decreasing mean z score, and some species with unacceptably large standard deviations were excluded. We also discarded a few arrayed sequences for which no cognate mRNAs had been identified.

shows those specifically immunoprecipitated mRNA species for which the mean relative Table Ia abundance was highest after applying this screen.

The dual-channel procedure was then used to compare the relative abundances of mRNA species in the immunoprecipitated vs. total polyA populations. The total population of polyA RNAs present in the input extract (before immunoprecipitation) was amplified and the

+ +
resulting Cy5-labeled transcripts were mixed with an aliquot of Cy3-labeled transcripts derived from the Sam68 immunoprecipitate. After hybridization to the array, z scores were calculated for each channel. The species of scored 0.6 to 1.0 units higher in the Table Ia immunoprecipitated than in the total population, consistent with their enrichment.

There was a discernible pattern in the biological functions of these mRNAs (

). When assessed by Gene Ontology annotation Fig. 1E for generic cellular process or component, more than 20 of the mRNAs (9 transcripts) were involved in intracellular or % receptor-associated signal transduction, as they coded for e.g. receptor subunits, protein kinases, protein phosphatases, and associated regulatory factors. Another 20 of the mRNAs coded for proteins involved in protein transport, either as regulators of vesicular traffic, or % of microtubule-dependent motion. These proportions are significantly higher than what would be expected based on the overall distribution of biological functions among the genes represented on the micro-array (

). By comparison, transcription regulators Fig. 1E appeared to be under-represented among the major Sam68-associated mRNAs. Assessment of the litterature further indicates that some of the proteins of contribute to exocytic and endosomal traffic (Arf-1, BRI-3, vacuolar ATPase) or to cytoskeletal regulation Table I (stathmin) event-though this is not apparent from their GO function label. All of these processes are heavily involved in the local response to stimuli which affect neuronal homeostasis and plasticity. Indeed, more than one third of the genes of code for proteins directly Table Ia involved in synaptic plasticity, connectivity development, or neurodegenerative disease (

). Table Ib The -actin mRNA, which was detected among the most abundant species in our screen, had formerly been detected by RT-PCR in β immunoprecipitates of brain Sam68 (

), and was known as a specific target for recombinant Sam68 ( ). Ben [START_REF] Fredj | Depolarization-induced translocation of the RNA-binding protein Sam68 to the dendrites of hippocampal neurons[END_REF][START_REF] Itoh | Identification of cellular mRNA targets for RNA-binding protein Sam68[END_REF] This provided a positive control for the immunoprecipitation procedure. The Gephyrin mRNA provided a negative control, as it was found neither by RT-PCR, nor in the micro-array analysis (data not shown and ( )). [START_REF] Grange | Somatodendritic localization and mRNA association of the splicing regulatory protein Sam68 in the hippocampus and cortex[END_REF] The most enriched mRNA, Eef1a1, coded for the ubiquitous isoform of translation elongation factor eEF1A, an important player in both protein synthesis and long-term synaptic change (see Discussion). The z score value of the Eef1a1 fluorescence indicates that the representation of Eef1a1 among immunoprecipitated mRNAs is significantly higher than average (p 0.004).

= RT-PCR with gene-specific primer pairs (see ) was used to confirm the specific coimmunoprecipitation of a range of mRNAs Table II detected in the micro-array analysis, either among the more enriched species listed in , or among mRNAs with lower relative Table Ia abundance. In addition to -actin, confirmed species included Eef1a1, Calmodulin I and Uch-L1 (

). Interestingly, the Arc/Arg 3.1 β Fig. 2 mRNA was present among the less abundant mRNAs in the micro-array analysis, and could also be detected by RT-PCR (see ). Fig. 2A Since both Arc and Eef1a1 are dendritic, activity-responsive mRNAs, this raises the possibility that upon exit to the somatodendritic compartment Sam68 may play a role in activity-induced translation of neuronal mRNAs.

As Eef1a1 was the most enriched of Sam68-associated mRNAs in our assay, and since its translational regulation by synaptic inputs is well-documented (see below), we decided to investigate further its relationship with Sam68. To reconstitute the interaction between Eef1a1 mRNA and Sam68 in a heterologous system, myc-tagged Sam68 or a control plasmid were transiently expressed in HEK 293 cells; cytoplasmic extracts prepared from the transfected cells were immunoprecipitated with anti-myc antibody, and the immmunoprecipitates were probed by RT-PCR using the Eef1a1-specific primer pair. Endogenous Eef1a1 transcripts were detected in anti-myc immunoprecipitates from myc-Sam68-expressing cells but not from control cells ( , lanes 1 6). This result was reproduced by Fig. 2C quantitative RT-PCR (

). Thus, the Eef1a1 mRNA associates with , overexpressed Sam68. By contrast, the -tubulin Fig. 2C bona fide β mRNA was absent from myc-Sam68 immunoprecipitates ( , lanes 7 12), supporting the mRNA sequence specificity of binding. Fig. 2B -

Identification of as a translationally activated mRNA in depolarized neurons Eef1a1

The Eef1a1 mRNA contains a 5 oligopyrimidine tract and belongs to the general group of signal-responsive mRNAs encoding ′ components of the translation apparatus (

). Moreover, translation of Eef1a1 is stimulated during synaptic Petroulakis and Wang 2002 change (( ). To determine whether Eef1a1 might belong to the population of translationally activated mRNAs in our [START_REF] Tsokas | Local Protein Synthesis Mediates a Rapid Increase in Dendritic Elongation Factor 1A after Induction of Late Long-Term Potentiation[END_REF] system of in vitro depolarised neurons, primary cultures of cortical neurons were subjected to control treatement or prolonged depolarisation, lysed under nondenaturing conditions, and the post-nuclear supernatants were fractionated on sucrose density gradients, as described by (

). Fractions were analysed for their RNA content by continuous monitoring of UV absorbance Krichevsky and Kosik 2001 during collection. Fractions corresponding to the free mRNPs, 80S monosomes, polyribosomes, and RNA granules (migrating down to the bottom of the gradient) were analysed by RT-PCR with Eef1a1-specific primers. In non-stimulated neurons, the Eef1a1 mRNA was associated to the same extent with free mRNPs and with polysomes. Upon depolarisation, Eef1a1 became nearly undetectable in the free mRNP fraction, and strongly accumulated in the polysome fraction (

). These data indicate that Eef1a1 mRNA translation is Fig. 3A activated by depolarisation. Consistent with this result, depolarisation of cultured cortical neurons induced a significant increase in the total level of eEF1A protein, as detected by Western immunoblotting (

).

Fig. 3B

Depolarisation-inducible association of Sam68 with polysomes

We previously reported that Sam68 was associated with polyribosomes in cytoplasmic extracts derived from adult rat cortical tissue ( ). To confirm that a similar association occurred in cultured cortical neurons, and to determine whether the interaction [START_REF] Grange | Somatodendritic localization and mRNA association of the splicing regulatory protein Sam68 in the hippocampus and cortex[END_REF] could be regulated by depolarisation, extracts were prepared and fractionated exactly as in . Fractions were then analysed by Fig. 3A Western immunoblotting with anti-Sam68 antibody. In extracts from control, quiescent neurons, Sam68 mostly comigrated with free mRNPs; a minor fraction sedimented in the RNA granule fraction ( , top). Following depolarisation, Sam68 accumulated in Fig. 3C fractions containing polysomes and granules, even though the total amount of RNA in those fractions was slightly lower than in the control condition ( , bottom). This result is consistent with the depolarisation-induced accumulation of Sam68 fluorescence in RNA-enriched Fig. 3 dendritic clusters, which we have previously reported (

). Thus, in depolarised neurons, the increase in Eef1a1 Ben [START_REF] Fredj | Depolarization-induced translocation of the RNA-binding protein Sam68 to the dendrites of hippocampal neurons[END_REF] translation correlates with the loading of Sam68 onto translated mRNAs.

Effect of dominant-negative Sam68 on neuronal eEF1A expression

The association of Sam68 to actively translated Eef1a1 mRNA suggests that Sam68 might regulate the synthesis of eEF1A. To investigate this point, we assayed the level of eEF1A immunoreactivity in cultured hippocampal neurons that expressed GFP-tagged derivatives of either wild-type Sam68 or Sam68 C, a C-terminally truncated Sam68 mutant. The latter mutant retains a significant affinity Δ for specific RNA sequences, but is deprived from tyrosine-rich and proline-rich motifs involved in protein-protein interactions with SH2 and SH3-containing ligands, and also from a C-terminal (nonclassical) nuclear localisation signal (

). In cultured cell lines, Sam68 Fig. 4F Δ C is constitutively cytoplasmic and displays transdominant-negative activity towards endogenous Sam68, when coexpressed with retroviral (HIV-1) mRNAs that require Sam68 for efficient utilization ( ; ). Sam68 C is specifically [START_REF] Reddy | Inhibition of HIV replication by dominant negative mutants of Sam68, a functional homolog of HIV-1[END_REF][START_REF] Soros | Inhibition of Human Immunodeficiency Virus Type 1 Rev Function by a Dominant-Negative Mutant of Sam68 through Sequestration of Unspliced RNA at Perinuclear Bundles[END_REF] Δ recruited to these viral mRNAs once they have reached the cytoplasm, and appears to block their normal interaction with the translation apparatus (

). We reasoned that expression of Sam68 C in neurons should similarly allow us to probe the contribution of [START_REF] Soros | Inhibition of Human Immunodeficiency Virus Type 1 Rev Function by a Dominant-Negative Mutant of Sam68 through Sequestration of Unspliced RNA at Perinuclear Bundles[END_REF] Δ endogenous Sam68 to the translational activity of target neuronal mRNAs, as distinct from its role in nuclear mRNA processing. In transfected neurons, the GFP-Sam68 C protein was mostly excluded from the neuronal nucleus and concentrated in clusters within the Δ cell body; it was also detectable in dendritic shafts (

). The relative expression level of endogenous eEF1A in transfected and Fig. 4, A2, B non-transfected neurons was evaluated by immunofluorescent staining with an anti-eEF1A monoclonal antibody. Among the neurons of a field, those expressing GFP-Sam68 C typically had a lower level of eEF1A immunoreactivity than surrounding, nontransfected neurons ( Δ ). This was not the case in neurons that had been transfected with GFP-tagged wild-type Sam68 (

). Neurons Δ immunofluorescence, whereas GFP-Sam68 did not ( ). As a control, GFP-Sam68 C did not affect the abundance of coexpressed Fig. 4E Δ mRFP. Since GFP-Sam68 C functions in the cytoplasm, and presumably competes with endogenous Sam68 for binding sites in Δ mRNA-protein complexes, this result suggests that endogenous Sam68 positively regulates the translation of eEF1A-encoding mRNAs.

Possible post-synaptic anchoring by Sam68

In non-neuronal cells, Sam68 is known to bind to membrane-associated signalling proteins (

). In neurons, [START_REF] Lukong | Sam68, the KH domain-containing superSTAR[END_REF] given its association with dendritic mRNAs that become actively translated in response to local synaptic stimuli ( ), we [START_REF] Tsokas | Local Protein Synthesis Mediates a Rapid Increase in Dendritic Elongation Factor 1A after Induction of Late Long-Term Potentiation[END_REF] wondered whether Sam68 might physically interact with synaptic receptors. In synaptic membranes prepared from rat cortex, both the NMDA receptor and the post-synaptic density protein PSD95 could be co-immunoprecipitated with Sam68 ( , lanes 1 6). By Fig. 5 contrast, AMPA receptors were essentially absent from these immunoprecipitates (lanes 7 9), consistent with the notion that Sam68 was specifically recruited by the NMDA receptor complex. We could also detect Sam68 in NMDA receptor immunoprecipitates (lanes 10 11).

-Thus Sam68 might conceivably provide a platform for docking specific neuronal mRNAs and/or for recruiting specific translation factors at subsynaptic sites.

Discussion

The present sudy indicates that brain Sam68 is associated with a wide but selective range of cytoplasmic mRNAs, among which transcripts encoding long-term plasticity related proteins are prominent. Sam68 might collectively regulate the synthesis of an entire group of such proteins, in an activity-dependent fashion. The set of Sam68-associated mRNAs we found in brain differs from those reported in non-neuronal cell lines or lineages ( ; ;

). Extensive comparison of the [START_REF] Itoh | Identification of cellular mRNA targets for RNA-binding protein Sam68[END_REF][START_REF] Paronetto | The nuclear RNA-binding protein Sam68 translocates to the cytoplasm and associates with the polysomes in mouse spermatocytes[END_REF][START_REF] Tremblay | mRNAs associated with the Sam68 RNA binding protein[END_REF] different sets is difficult, since the screening methods were different. However, two of the mRNAs, -actin and dynein, were found in both β the study by ( ) and our screen, providing a reliable positive control. Taken together, the various screens suggest that [START_REF] Itoh | Identification of cellular mRNA targets for RNA-binding protein Sam68[END_REF] mRNA recognition by Sam68 is highly dependent on cellular context.

One of most enriched among the Sam68-associated cortical transcripts was the Eef1a1 mRNA. Although Eef1a1 is ubiquitous, the Sam68-Eef1a1 mRNA interaction was apparently not detected in the previous studies. This may be due to the sensitivity of the micro-array sampling procedure; alternatively, the interaction may be favoured by neuron-specific factors. However, it was possible to reconstitute the Sam68-Eef1a1 mRNA interaction in transfected HEK cells by using overexpressed, myc-tagged Sam68. The interaction is phylogenetically conserved since HEK 293 cells are of human origin; this is consistent with the >90 identity of human and rat Eef1a1 % nucleotide sequences, including in the 3 untranslated region. Interestingly, based on free energy minimization, the entire 3 UTR of the

′ ′
Eef1a1 mRNA is predicted to fold into a single, long hairpin displaying at its extremity a U-rich terminal loop ( ). This is reminiscent Fig. 6 of the U6 motif-containing stem-loop structure that mediates recognition of the -actin mRNA by Sam68 ( ).

β in vitro [START_REF] Itoh | Identification of cellular mRNA targets for RNA-binding protein Sam68[END_REF] However, our experiments do not rule out the possibility that the Sam68 Eef1a1 mRNA association may be mediated by a third molecule. Further studies will be required to establish whether the 3 UTR structure forms a direct binding site for Sam68 on the Eef1a1 ′ mRNA.

In dissociated cortical neurons, chemically induced depolarisation is known to generate bursts of action potentials and to recruit some of the pathways that underlie synaptic plasticity. While causing the translational repression of multiple mRNA species, depolarisation activates the translation of a select group of transcripts ( ). Our data indicate that Eef1a1 belongs to this group. [START_REF] Krichevsky | Neuronal RNA granules: a link between RNA localization and stimulation-dependent translation[END_REF] Eef1a1 encodes the ubiquitous isoform of translation elongation factor eEF1A, which transports amino-acyl-tRNA to the A site of the ribosome during protein synthesis. In addition, eEF1A also anchors mRNAs to the actin cytoskeleton, and can bundle actin filaments according to a unique spatial arrangement (

). Local provision of eEF1A may play a role in both biosynthetic and structural Liu et al. 2002a aspects of synaptic plasticity, and perhaps establish a link between these two facets of long-term synaptic change. In , synthesis of Aplysia the eEF1A homologue is instrumental to maintenance of synapse-specific long-term facilitation (

). In the rodent [START_REF] Giustetto | Axonal transport of eukaryotic translation elongation factor 1a mRNA couples transcription in the nucleus to long-term facilitation at the synapse[END_REF] hippocampus, translation of the Eef1a1 mRNA is regulated by synaptic signals in complex ways, which appear to differ between preparations. In hippocampal slices from young rats, Eef1a1 translation has been shown to be rapidly induced during tetanus-induced LTP of CA3-CA1 synapses, subject to activation of the rapamycin-sensitive mTOR/S6 Kinase pathway (

). This finding is [START_REF] Tsokas | Local Protein Synthesis Mediates a Rapid Increase in Dendritic Elongation Factor 1A after Induction of Late Long-Term Potentiation[END_REF] consistent with the fact that the Eef1a1 mRNA contains a 5 oligopyrimidine tract, which is a signature for mTOR-inducible mRNAs.

′

Interestingly, in adult rat hippocampus , LTP of dentate granule synapses induced redistribution rather than neosynthesis of eEF1A in vivo protein. In contrast, long-term depression elicited by pharmacological stimulation of metabotropic glutamate receptors (mGluRs) was accompanied by a large increase in eEF1A protein, without change in Eef1a1 mRNA level (

). Thus, the induction of [START_REF] Huang | The mRNA for Elongation Factor 1 Is Localized in Dendrites and Translated in Response to Treatments That Induce Long-Term α Depression[END_REF] eEF1A synthesis seems to be exquisitely sensitive to the spatio-temporal and cell-specific details of synaptic signalling, suggesting that multiple effectors regulate the translation of Eef1a1 in neurons. One of these effectors may be the RNA-binding protein FMRP, since in vitro and in cell lines, FMRP has been reported to bind and repress the Eef1a1 mRNA ( ); furthermore it is tempting to [START_REF] Sung | The fragile X mental retardation protein FMRP binds elongation factor 1A mRNA and negatively regulates its translation in vivo[END_REF] speculate that removal of this putative inhibition underlies the effect of mGluRs on Eef1a1 translation (

). The present data [START_REF] Bear | The mGluR theory of fragile X mental retardation[END_REF] suggest that Sam68 is also part of the regulatory machinery that controls neuronal Eef1a1 translation, with an action distinct from that of FMRP. Since depolarisation increased the recruitment of Sam68 to polysomal mRNAs, it is likely that Eef1a1 transcripts bound to Sam68 were engaged in translation. The finding that the dominant-negative GFP-Sam68 C, but not GFP-Sam68(WT) inhibited eEF1A protein Δ accumulation suggests that endogenous Sam68 may be required for the translational activation of Eef1a1 mRNA. Since the dominant-negative effect of GFP-Sam68 C was already detected in unstimulated neurons, the low amount of Sam68 existing in the Δ cytoplasm prior to depolarisation may contribute to generating the proportion of actively translated Eef1a1 mRNA seen under basal conditions. In line with this possibility, Sam68 has been reported to interact with RNA helicase A and with the mRNA export factor Tap ( depolarisation, the largely enhanced export of Sam68 to the cytoplasm may cooperate with the action of signalling cascades such as the mTOR pathway for translational stimulation of activity-responsive mRNAs exemplified by Eef1a1. A similar scheme may apply to certain non-neuronal systems; in the case of meiotic male gametes, recently confirmed the occurrence of signal-induced [START_REF] Paronetto | The nuclear RNA-binding protein Sam68 translocates to the cytoplasm and associates with the polysomes in mouse spermatocytes[END_REF] transfer of Sam68 to the cytoplasm and its binding to polysomes.

Ackowledgements:

INSERM; Universit Joseph Fourier; Minist re de l Enseignement Sup rieur et de la Recherche; Association pour la Work supported by Functional profile of the 43 mRNAs displaying the highest mean z scores in the E.

immunoprecipitates (see

). For each transcript, a generic function was registered (identified by Gene Ontology descriptors of the Table Ia mRNA in the NIH database; see ). Black barss: number of occurrences of the indicated functional descriptors in the 43 mRNA Table Ia sample. Empty bars: expected number of occurrences of the descriptors, based on their frequencies among mRNAs of the micro-array. The difference is highly significant (p<0.0001, chi-square test), indicating enrichment for specific functional categories.

Fig. 2

Validation of specific micro-array hybridization results by RT-PCR. Cortical tissue extract was subjected to immunoprecipitation with A.

anti-Sam68 ( IP ) or preimmune ( Pre ) IgG as shown; total mRNA was purified from the immunoprecipitates, and subjected to reverse " "

" "

transcription and amplification by PCR, using the indicated gene-specific primers ( ). The PCR products were visualized by Table II fluorescent staining with ethidium bromide. The expected sizes of PCR products are indicated (Eef1a1: 336 bp; Uch-L1: 187 bp; -actin: 178 β bp; Calm-1: 229 bp; Arc: 287 bp ). Positive controls for the amplification reactions were obtained by performing PCR with total mRNA ( Tot " "

) directly derived from the input extract. No amplification took place without cDNA ( None or 0 ).

" " " "

Reconstitution of the association B.

between Sam68 and the Eef1a1 mRNA in cultured cells. HEK 293 cells were transfected with a myc-tagged Sam68 expression construct, or with control vector, as indicated. Cytoplasmic extracts were prepared and immunoprecipitated with anti-myc antibody, and subjected to RT-PCR with Eef1a1 (lanes 1, 3) or with -tubulin (lanes 7,9) primers. As positive controls for amplification, RT-PCR products were β generated with total mRNA from the transfected cells (lanes 2, 4, 8, 10) or from rat cortex (lane 5, 11). Lanes 6, 12: negative control for PCR.

The amounts of endogenous Eef1a1 mRNA in anti-myc immunoprecipitates from the indicated transfections were determined by C.

quantitative ( real-time ) PCR (n 3 for each transfection).

, arbitrary units.

" " = a.u.

Fig. 3

Depolarisation induces an increase in Eef1a1 mRNA translation. Cultured cortical neurons were incubated for 6 h in 5mM or 25 mM KCl, A.

then cytoplasmic extracts were prepared and equal protein amounts were subjected to fractionation on sucrose density gradients. For each gradient, representative fractions corresponding to translationally inactive mRNPs, 80S monosomes, polysomes, and RNA granules were analysed by RT-PCR with specific primers to amplify the Eef1a1 mRNA. The PCR products were analysed side by side on an agarose gel ("-" and lanes: gradient fractions from control and depolarised neurons; respectively) and visualized by fluorescent staining with ethidium "+"

bromide. Greyscale was inverted to improve contrast. Depolarisation increases the amount of eEF1A protein. Cortical neurons were treated B.

as in A, then lysed, and equal amounts of total protein were analysed by SDS-polyacrylamide gel electrophoresis and immunoblotting. Left:

the same blot was probed first with anti-eEF1A monoclonal antibody, then with anti-actin antibody. Right: the relative amount of eEF1A in each lane was measured by densitometric scanning, and normalized to the amount of actin in the same lane to correct for gel loading error.

The histogram shows the average ( / S.D.) of normalized eEF1A amount in each condition.

17 amplified

 17 by using the gene-specific primer pairs listed in . For classical PCR, after 2 min at 95 C, 30 cycles were performed (15 s TableII

  °at 95 C, 30 s annealing, and 30 s at 72 C for each cycle). For quantitative PCR by real-time fluorometry (Light Cycler, Roche Molecular °°Systems), DNA was denatured at 95 C for 5 min, then subjected to ~35 40 amplification cycles (15 s at 95 C, 5 s annealing, 15 s at 72 C, °-°°all temperature changes at 20 C/s). Annealing took place at 55 C except for Arc (57 C). The relative amounts of specific DNA template °°°w ere determined by counting the number of cycles needed to reach the inflection point ( crossing point at which d DNA /dt 0) of the

  Housing and handling of animals complied al. 1998 with the regulations issued by the European Union and INSERM and were approved by the local veterinary authorities. The GFP-Sam68 (WT) and GFP-Sam68 C plasmids were kind gifts from S. Richard (McGill University, Montr al, Canada). The mRFP1 plasmid Δ é

  Fig.4, A1, C3 some general toxicity of the construct. The effect of GFP-Sam68 C expression on endogenous eEF1A immunofluorescence was Δ quantitated by using as internal reference value the average immunofluorescence of nontransfected neurons surrounding the transfected cells (see Materials and Methods). This quantitation procedure indicated that GFP-Sam68 C reproducibly lowered the level of eEF1A

)

  , two proteins known to promote translation of specific viral and cellular mRNAs ( ; ). Reddy et al. 2000 Hartman et al. 2006 Li et al. 2006 Kinases recruited by the C terminus of Sam68 might also be required to phosphorylate and release mRNA-bound translation inhibitory factors, similar to the case of hnRNP-K phosphoylation by Src ( ). GFP-Sam68 C, which is defective for Ostareck-Lederer et al. 2002 Δ certain protein-protein interactions, may displace the endogenous Sam68 and associated translation factors out of the target mRNA; indeed, in the case of viral mRNAs, GFP-Sam68 C functionally antagonizes RNA helicase A and Tap (

  Recherche contre le Cancer (ARC); Fondation pour la Recherche M dicale. é We thank Michel Volovitch and Alain Prochiantz for their kind cooperation in the use of micro-array analysis; St phane Richard and Roger é Y. Tsien for the generous gift of the GFP-Sam68 C and mRFP1 plasmids, respectively; Isabelle Barlat and Bruno Toqu for the kind gift of Δ é myc-Sam68 plasmid; and Michael Kiebler for discussions and reagents.

Fig. 1

 1 Fig. 1Scheme of the RNA-protein coimmunoprecipitation procedure, and image of a representative hybridization experiment. Green channel: A.Cy5-labelled probes obtained by amplification of polyA mRNA immunoprecipitated with the anti-Sam68 antibody. Red channel: + Cy3-labelled probes obtained after immunoprecipitation with control (preimmune) antibody. Distribution of paired fluorescence values B. obtained in A. X-axis: specifically immunoprecipitated transcripts. Y-axis: adventitious transcripts. A large number of transcripts are more abundant in the specific immunoprecipitate than in the control. Within-sample reproducibility of z score measurements. The results of two C. different hybridization experiments performed with the same sample are compared. The regression line is shown in red. Between-sample D. reproducibility of z scores. The results of hybridization experiments performed on two different brain extracts are compared. The regression line is shown in red. Note that mRNAs with highest values in either sample (to the right of the vertical green line and above the horizontal green line) are mostly confined to the top right quadrant.Functional profile of the 43 mRNAs displaying the highest mean z scores in the E.

  Sam68 with polysomal RNA and RNA granules. Cytoplasmic extracts from control and depolarised neurons were prepared and fractionated as in . The fractions were analysed by immunoblotting with anti-Sam68 antibody (left). The distribution of RNA along the gradient was A determined by continuous monitoring of UV absorbance at 254 nm during collection of the fractions (right). The various types of ribonucleoproteic assemblies are indicated.

Fig. 4

 4 Fig. 4Effect of dominant-negative Sam68 on eEF1A expression in cultured hippocampal neurons.The GFP-Sam68 (WT) and GFP-Sam68 C A-C: Δ proteins were expressed in hippocampal neurons, together with monomeric Red Fluorescent Protein (mRFP) as a morphological marker. Five days later (at 14 DIV), the neurons were fixed and stained by immunofluorescence with anti-eEF1A antibody; mRFP, GFP-Sam68 and endogenous eEF1A were simultaneously visualized by triple-colour fluorescence microscopy.close-up view of the three fluorescent A1-A3: markers in a GFP-Sam68 C-transfected neuron and a nearby, untransfected neuron (wide-field microscopy).: Note the lack of eEF1A Δ

Fig. 5

 5 Fig. 5Binding of Sam68 to the post-synaptic NMDA receptor complex. Purified synaptic membranes were solubilized under non-denaturing conditions, and subjected to immunoprecipitation with the indicated antibodies. Lanes 1, 4, 7: aliquot (10 ) of input lysate. After detecting % PSD-95, the blot of lanes 4 6 was stripped and reprobed to detect GluR2/3 subunits of AMPA receptors, shown in lanes 7 9. Lane 10: control -immunoprecipitation with a mouse monoclonal IgG, class-matched to the anti-NR1 antibody.

Table 1 Table Ia . Identity and generic function of major Sam68-associated mRNAs. ID: Genbank identifier of cloned sequence on the NIA 15 K micro-array. Name: name of transcript homologous to the sequence on the array (available through ). Mean z score and its standard deviation (n 3) were calculated for each transcript as described in the text. P values were calculated from z scores, http://lgsun.grc.nia.nih.gov/cDNA/15k.html = assuming normal distribution of scores. Biological process or component associated with transcripts were identified by reporting Gene Ontology (GO) descriptors used to annotate the transcripts in the NIA database (available through ). Rightmost column: identification of the GO descriptors by their numbering in the Gene http://lgsun.grc.nia.nih.gov/cDNA/NIA-CloneSet-GoTermFinder.html Ontology database.

 1Ia

	ID	Name	Mean z S.D. ±	p value Process or component	GO
	AW557547	Translation elongation factor 1 alpha 1, Eef1a1	2.66 0.97 ±	0.004 protein biosynthesis	06412
	BG086025	ATPase, H transporting, V1 subunit B, isoform 2, Atp6v1b2 +	2.55 1.07 ±	0.005 proton transport	15992
	BG076172	Calmodulin-1, Calm1	2.36 1.11 ±	0.009 signal transduction	07165
	BG088462	Zfhx1a, C2-H2 zinc finger protein homeobox 1a	2.35 1.29 ±	0.009 regulation of transcription	06355
	AW558451	Reticulon 3, Rtn3	2.28 0.60 ±	0.011 protein transport	15031
	BG088310	Prosaposin	2.1 0.88 ±	0.018 metabolism	08152
	BG071424	BRI3	2.04 0.28 ±	0.021 integral to membrane	16021
	BG073409	Stathmin 1, Stmn1	1.98 0.48 ±	0.024 signal transduction	07165
	BG065012	14-3-3 eta, Ywhah	1.86 0.82 ±	0.031 signal transduction	07165
	BG075901	Gamma-aminobutyric acid receptor subunit, Gabra3	1.86 0.36 ±	0.031 synaptic transmission	07268
	BG076067	Inositol hexaphosphate kinase 1	1.86 0.50 ±	0.032 metabolism	08152
	BG085717	14-3-3 epsilon, Ywhae	1.85 0.84 ±	0.032 signal transduction	07165
	BG082675	ADP-ribosylation factor 1, Arf-1	1.85 0.90 ±	0.032 signal transduction	07165
	BG075782	Protein phosphatase 1, catalytic subunit, gamma, Ppp1cc	1.83 0.76 ±	0.034 signal transduction	07165
	BG075243	Zinc transporter ZnT-3	1.8 0.27 ±	0.036 cation transport	06812
	BG077677	Beta-actin	1.78 0.53 ±	0.037 cytoskeleton	07010
	BG065113	Branched chain aminotransferase, bcat-1	1.78 1.51 ±	0.038 metabolism	08152
	BG064914	Malate dehydrogenase, Mor2	1.73 0.61 ±	0.042 metabolism	08152
	BG087365	Neuroplastin	1.69 0.86 ±	0.046 surface receptor signal transduction	07166
	BG073370	Tetratricopeptide repeat domain 3, Ttc3	1.69 1.29 ±	0.046 protein binding	05515
	BG074109	Heat shock protein 1, alpha, Hspca	1.67 1.15 ±	0.047 chaperone activity	03754
	BG088451	Tissue inhibitor of metalloproteinase 2, Timp2	1.61 0.77 ±	0.053 extracellular matrix	05578
	BG075319	Mitogen activated protein kinase 3, pp44 MAPK, Erk1	1.61 0.57 ±	0.054 signal transduction	07165
	BG074398	Matrix glycoprotein Sparcl1/hevin	1.54 0.64 ±	0.062 extracellular matrix	05578
	BG073255	Tetraspanin TM4-A	1.5 0.86 ±	0.066 integral to membrane	16021
	BG082737	Proline-arginine-rich end leucine-rich repeat protein, Prelp	1.46 0.64 ±	0.073 extracellular matrix	05578
	BG073185	BTB (POZ) domain containing 4, KBTBD4	1.41 1.05 ±	0.079 regulation of transcription	06355
	BG088334	Vacuolar protein sorting 41	1.4 0.05 ±	0.081 protein transport	15031
	BG087861	Dynein, cytoplasmic, intermediate chain 1, Dncic1	1.39 0.39 ±	0.082 microtubule-based movement	07018
	BG085988	Heterogeneous nuclear ribonucleoprotein H2, Hnrph2	1.35 0.91 ±	0.089 mRNA processing	06397
	BG073339	Splicing factor, arginine/serine-rich 5, Srp40/HRS	1.31 0.72 ±	0.096 mRNA processing	06397
	AW553322	G protein-coupled receptor associated sorting protein 1	1.27 0.86

±

0.102 protein transport

Table II

PCR primers used in this study.

[START_REF] Sung | The fragile X mental retardation protein FMRP binds elongation factor 1A mRNA and negatively regulates its translation in vivo[END_REF]

[START_REF] Sung | The fragile X mental retardation protein FMRP binds elongation factor 1A mRNA and negatively regulates its translation in vivo[END_REF]

[START_REF] Sung | The fragile X mental retardation protein FMRP binds elongation factor 1A mRNA and negatively regulates its translation in vivo[END_REF]

[START_REF] Sung | The fragile X mental retardation protein FMRP binds elongation factor 1A mRNA and negatively regulates its translation in vivo[END_REF]

[START_REF] Sung | The fragile X mental retardation protein FMRP binds elongation factor 1A mRNA and negatively regulates its translation in vivo[END_REF]

[START_REF] Sung | The fragile X mental retardation protein FMRP binds elongation factor 1A mRNA and negatively regulates its translation in vivo[END_REF]

[START_REF] Sung | The fragile X mental retardation protein FMRP binds elongation factor 1A mRNA and negatively regulates its translation in vivo[END_REF]

[START_REF] Sung | The fragile X mental retardation protein FMRP binds elongation factor 1A mRNA and negatively regulates its translation in vivo[END_REF]

[START_REF] Sung | The fragile X mental retardation protein FMRP binds elongation factor 1A mRNA and negatively regulates its translation in vivo[END_REF]

[START_REF] Sung | The fragile X mental retardation protein FMRP binds elongation factor 1A mRNA and negatively regulates its translation in vivo[END_REF]

[START_REF] Sung | The fragile X mental retardation protein FMRP binds elongation factor 1A mRNA and negatively regulates its translation in vivo[END_REF]

[START_REF] Sung | The fragile X mental retardation protein FMRP binds elongation factor 1A mRNA and negatively regulates its translation in vivo[END_REF]


Agnès Belly

Françoise Moreau-Gachelin

Rémy Sadoul

Yves Goldberg
email: yves.goldberg@ujf-grenoble.fr

Delocalization of the multifunctional RNA splicing factor TLS/FUS in hippocampal neurones: exclusion from the nucleus and accumulation in dendritic granules and spine heads

Keywords: dendritic mRNAs, neuronal mRNA traffic, RNA-binding protein, neuronespecific splicing

Delocalization of the multifunctional RNA splicing factor TLS/FUS in hippocampal neurones: exclusion from the nucleus and accumulation in dendritic granules and spine heads.

Introduction

The development and plasticity of synapses depend in part on protein synthesis taking place in the dendrites [START_REF] Steward | Compartmentalized synthesis and degradation of proteins in neurons[END_REF]. Polyribosomes are often docked at the basis of dendritic spines, and transferred into spine heads following long-term potentiation. It is largely unknown how such a synapse-specific regulation of mRNAs is achieved.

In cultured neurones, dendritic mRNAs such as CaMKIIα and Arc are packaged in clusters, termed "RNA granules", that move along the dendrites and sometimes locate close to synapses [START_REF] Rook | CaMKIIalpha 3' Untranslated Region-Directed mRNA Translocation in Living Neurons: Visualization by GFP Linkage[END_REF]. A growing number of RNA-binding proteins have been found to colocalize with RNA granules in situ, or to copurify with biochemically isolated granule preparations. A few of these proteins, including ZBP1 and FMRP, were shown to enter (and exit) spine heads [START_REF] Antar | Metabotropic Glutamate Receptor Activation Regulates Fragile X Mental Retardation Protein and Fmr1 mRNA Localization Differentially in Dendrites and at Synapses[END_REF]. A range of RNA-associated proteins, including translation factors and some RNA granule proteins, have also been identified in highly purified postsynaptic density preparations, consistent with synaptic localization of ribonucleoproteic complexes [START_REF] Peng | Semiquantitative proteomic analysis of rat forebrain postsynaptic density fractions by mass spectrometry[END_REF]. The molecular interactions responsible for retaining such complexes in the spine are undefined; they may involve anchoring of RNA-associated proteins to postsynaptic density proteins.

A few years ago, in a large-scale analysis of proteins that copurified with NMDA receptor from mouse brain extracts, the RNA-binding protein TLS (also called FUS) was found to exist in the receptor complex [9]. TLS (Translocated in Liposarcoma) has so far been known for its manifold functions in nuclear processes, including DNA transcription, pre-mRNA processing, homologous recombination, and DNA repair. The biological importance of the protein is underscored by the rapid postnatal death of TLS-deficient mice [START_REF] Hicks | Fus deficiency in mice results in defective B-lymphocyte development and activation, high levels of chromosomal instability and perinatal death[END_REF] and by the primary role of TLS or TLS-derived gene fusions in the evolution of specific tumours [START_REF] Perrotti | TLS/FUS, a pro-oncogene involved in multiple chromosomal translocations, is a novel regulator of BCR/ABL-mediated leukemogenesis[END_REF].

TLS interacts with general and promoter-specific transcription factors and with elements of the splicing machinery [6 , 10]. Consistent with its known function, in proliferating cells, TLS is concentrated in specific regions of the cell nucleus; however, TLS is capable of shuttling inserm-00451275, version 1 -28 Jan 2010 between nucleus and cytoplasm, a minor fraction of TLS molecules being associated with cytoplasmic mRNAs [START_REF] Zinszner | TLS (FUS) binds RNA in vivo and engages in nucleo-cytoplasmic shuttling[END_REF].

The identification of TLS as a (direct or indirect) partner of the NMDA receptor raises the possibility that this protein might participate in the docking and/or regulation of mRNAs at excitatory synaptic sites. However, it has not yet been established whether the ordinarily nuclear TLS can be detectably localized at synaptic or dendritic sites, and whether the protein colocalizes with NMDA receptor clusters in defined regions of the brain. This question is addressed here.

inserm-00451275, version 1 -28 Jan 2010

Materials and Methods

Antibodies and plasmid

The anti-TLS rabbit antiserum has been described [START_REF] Hallier | The Transcription Factor Spi-1/PU.1 Interacts with the Potential Splicing Factor TLS[END_REF]; this antibody does not cross-react with the TLS-related protein EWS (unpublished data). Affinity-purified anti-TLS IgG (used in all experiments) was purified from the antiserum as described [START_REF] Harlow | Using Antibodies: A laboratory Manual[END_REF], using beads that had been coupled to a GST-TLS fusion protein [START_REF] Hallier | The Transcription Factor Spi-1/PU.1 Interacts with the Potential Splicing Factor TLS[END_REF]; the antibody was further depleted of any anti-GST components by adsorption to unfused GST. The anti-Sam68 monoclonal antibody was derived from either clone 7-1 (Santa Cruz, Inc), or clone P20120 (BD Transduction). The monoclonal antibody raised against the myc epitope (clone 9E10) was obtained from Santa Cruz, Inc. Horseradish peroxidase (HRP)-conjugated protein A was obtained from BD Transduction. HRP-conjugated goat anti-mouse antibody was purchased from Jackson Laboratories. Fluorescent conjugates were purchased from Molecular Probes. The myc-TLS plasmid (pcs3-mt-TLS) [START_REF] Hallier | The Transcription Factor Spi-1/PU.1 Interacts with the Potential Splicing Factor TLS[END_REF]allows expression of full-length TLS fused in-frame with a hexamer of the myc epitope at the N terminus.

Fractionation of hippocampal tissue

All procedures using animals conformed to institutional guidelines, in compliance with European Community Council Directive 86/609. Hippocampi were dissected from adult (200-210 g) male OFA rats (Charles Rivers, Lyon, France), and subjected to subcellular fractionation essentially as described by Feng et al. [START_REF] Feng | Fragile X Mental Retardation Protein: Nucleocytoplasmic Shuttling and Association with Somatodendritic Ribosomes[END_REF]. The hippocampi were manually ground in a Potter homogenizer with a Teflon pestle (0.15 mm clearance, 15 strokes), in 1 ml ice-cold H buffer (4mM Hepes-NaOH, pH 7.4, 0.32 M sucrose, 1 mM MgCl 2 , 1 mM sodium orthovanadate, 1 mM dithiothreitol, 100 nM microcystine-LR (Sigma), protease inhibitors (EDTA-free "Complete", Roche Molecular). The homogenate was filtered through a 100 µmmesh nylon filter (Millipore) and centrifuged at 800 x g for 10 min. at 4°C. The resulting S1 supernatant was centrifuged at 9500 x g for 20 min. at 4°C. The S2 supernatant was inserm-00451275, version 1 -28 Jan 2010 centrifuged at 130,000 x g for 1 hr at 4°C. For all fractions, total protein concentration was measured and samples were boiled for 5 min. in Laemmli gel buffer. Equal protein amounts of the samples were resolved on a 8% polyacrylamide-SDS gel and transferred onto a PVDF membrane (Millipore). The membrane was probed with anti-TLS antibody (200 ng/ml) or anti-Sam68 antibody (200 ng/ml), and after incubation with HRP-conjugated secondary reagents, bands were revealed by chemiluminescence (Super-Signal Pico, Pierce).

Photographs of the blots were digitized with a flatbed scanner. Care was taken to ensure signal linearity.

Immunostaining of brain tissue

Vibratome sections (30 µm thick) of PFA-fixed adult rat hippocampus were prepared as previously described [START_REF] Grange | Somatodendritic localization and mRNA association of the splicing regulatory protein Sam68 in the hippocampus and cortex[END_REF]. For staining, the sections were incubated at 4°C for 20 min in cold (-20°C) ethanol; rinsed with TBS (20 mM Tris-HCl, pH 7.4, 150 mM NaCl); preincubated for 30 min at room temperature in blocking buffer (TBS containing 3% goat serum and 0.1% Triton X-100); and incubated with primary antibodies (2 µg/ml) in blocking buffer for 48 hr at 4°C. Sections were then washed (3x10 min in TBS containing 0.1% Triton X-100); incubated for 1 hr at R.T. with blocking buffer containing Alexa 488-conjugated goat antirabbit IgG and Alexa 594-conjugated goat anti-mouse IgG; washed as above; rinsed in water, and mounted on slides in Mowiol medium.

Immunostaining of cultured neurones

Hippocampal neuronal cultures were prepared from E19 rat embryos (Charles Rivers) essentially as described by Banker and Goslin [START_REF] Banker | Rat Hippocampal Neurons in Low-Density Culture[END_REF] with a few modifications. After dissecting, trypsinizing and triturating the hippocampi, hippocampal neurones were resuspended in Dulbecco's minimal essential medium containing glutamine, penicilline, streptomycine, and 10% heat-inactivated horse serum (Invitrogen), and plated at a density of 0.5 -1 x 10 4 per cm 2 on 13 mm glass coverslips (Marienfeld, Germany) coated with poly-L-lysine (Sigma).

inserm-00451275, version 1 -28 Jan 2010

Supporting high-density cultures were prepared by seeding in the same medium 1.5 x 10 5 hippocampal cells per well in 24-well plates (Becton-Dickinson) in which small pegs had been formed at the bottom of the wells, by brief contact with a soldering iron. After 3-4 hours, the medium of the wells was changed to Neurobasal (Invitrogen) containing antibiotics, glutamine, sodium pyruvate and B27 supplement (Invitrogen). The coverslips were transferred into the wells, with attached neurones facing downward. The cultures were fed once a week with 1:3 volume of fresh medium. After 4 weeks, neurones were fixed for 20 min. at R.T. in PBS containing 4% paraformaldehyde and 4% sucrose; washed with PBS; permeabilized for 10 min. at 4°C with 0.1% Triton X-100 in PBS; preincubated for 1 hr in PBS containing 3% bovine serum albumin (BSA); and stained for 1 hr in PBS containing 3% BSA and 2 µg/ml primary antibodies. After rinsing in PBS, fluorescent conjugates were applied for 1 hr (Alexa 488-conjugated anti-rabbit IgG, rhodamine-conjugated phalloidin, and (for triple labelling) Alexa 350-conjugated anti-mouse IgG in PBS/ 3% BSA). Coverslips were rinsed 3 times in PBS, 3 times in water, and mounted on slides in Mowiol medium.

Microscopy

Images of the brain sections and of doubly stained neurones were acquired as z-stacks of 512

x 512 pixel frames with a Zeiss LSM 510 laser scanning confocal microscope, using a 63X (1.4 NA) objective. For dual colour imaging, care was taken to exclude contamination between channels, and to maximize the dynamic range at both wavelengths. Images of triply stained neurones were acquired as 1300 x 1030 pixel frames with a Zeiss Axiovert 200 microscope, using a 100x (1.4 NA) objective, appropriate bandpass filters (Chroma Technologies) and a cooled CCD camera (Micromax, Roper Scientific). Again, care was to taken to maximize the dynamic range of all channels. Image files were processed with Metamorph (Universal Imaging) and Photoshop (Adobe). Non-specific background fluorescence, estimated as at most (mean+2SD) of pixels in an empty region, was subtracted inserm-00451275, version 1 -28 Jan 2010 from the raw images; the resulting images were contrasted to highlight the somatodendritic granules. To establish the specificity of TLS labelling, images of specifically stained and control sections were acquired under identical illumination and scanning conditions, and the average fluorescence of the control section was used as background value for both images.

Culture, transfection and staining of HEK-293 cells

HEK-293 cells were maintained in DMEM supplemented with 10% fetal calf serum (Invitrogen). The cells were seeded on glass coverslips kept in a 6-cm dish, and transfected the next day with 5 µg myc-TLS plasmid using Fugene reagent (Roche Molecular). 48 hr later cells were fixed in PBS containing 4% paraformaldehyde for 20 min. at R.T., and subjected to immunofluorescent staining essentially as described for neurones, using 2 µg/ml anti-TLS antibody and 200 ng/ml anti-myc monoclonal antibody.

inserm-00451275, version 1 -28 Jan 2010

Results

Adult hippocampal tissue was fractionated into nuclear, membrane and cytosolic fractions, and the relative abundance of the TLS protein was determined by Western immunoblotting of the various fractions. Unlike the case in proliferating cell types, TLS was nearly absent from the nuclei (Fig. 1A, upper panel, lane 2). The 75 kD band expected for TLS (arrow) was enriched in the soluble fraction (lane 6). Some of the 75 kD protein could also be recovered together with microsomal membranes and polysomes in the P3 pellet (lane 5).

As a control for the integrity of the various fractions, the blot was reprobed with an antibody raised against Sam68, an RNA-binding protein of a different type. Consistent with known results [START_REF] Grange | Somatodendritic localization and mRNA association of the splicing regulatory protein Sam68 in the hippocampus and cortex[END_REF], Sam68 was most abundant in the nuclear fraction (lower panel). Thus, the absence of nuclear TLS did not arise from mere breakdown of the nuclei. This result suggests that in hippocampal tissue, most of the TLS protein resides in the cytoplasmic compartment, with a minor but definite fraction associated with the polysomal fraction.

In addition, an immunoreactive band of 100 kD was reproducibly detected, and enriched in the P2 and P3 fractions (arrowhead). Whether this band arises from antibody cross-reactivity or represents some modified form of the TLS protein is not known at present.

To define the subcellular localization of the TLS protein in more detail, immunofluorescent detection was performed. The specificity of our anti-TLS antibody in immunofluorescence assays was first verified in cultured HEK293 cells that had been transfected with a myc-TLS construct. The myc-tagged protein was detected by dual staining with anti-myc monoclonal antibody and anti-TLS antibody. In cells expressing the construct, the anti-TLS antibody generated intense nuclear staining that exactly overlapped with the distribution of the myctagged protein (Fig. 2A). In addition, in cells that had failed to take up the myc-TLS plasmid, the anti-TLS antibody revealed an identical but much fainter pattern of nuclear staining, corresponding to the usual distribution of the endogenous TLS protein in proliferating cells inserm-00451275, version 1 -28 Jan 2010 (Fig. 2B, arrow). Thus, the anti-TLS antibody used in this study is capable of specifically staining the bona fide TLS protein. No staining was obtained with preimmune IgG (not shown).

The distribution of TLS staining in sections of adult hippocampus was then examined by confocal microscopy and compared to that of two reference proteins, i.e. the predominantly nuclear RNA-binding protein Sam68, and the NR1 subunit of the NMDA receptor. TLS staining was detected in the cell bodies and throughout the dendritic layers (stratum moleculare, stratum radiatum), but was largely excluded from the neuronal nuclei (Fig. 2C).

In contrast, the pattern of Sam68 staining was predominantly nuclear and perinuclear, with a low level of dendritic fluorescence, in agreement with our previous results (Fig. 2D). Higher magnification views indicated that the TLS immunofluorescence displayed both a granular component and a diffuse component. This pattern agrees well with the distribution found by biochemical analysis. Individual TLS clusters could be visualized in the perinuclear region of dentate granule neurones (Fig. 2F) and in the perikaryon and dendritic trunk of CA1 pyramidal neurones (Fig. 2I). Despite the overall clearly distinct distribution of the two proteins, some of the TLS clusters overlapped with somatodendritic Sam68 granules (arrows in Figs. 2F-H, I-K). Because Sam68 granules have been shown to associate with RNA [START_REF] Ben Fredj | Depolarization-induced translocation of the RNA-binding protein Sam68 to the dendrites of hippocampal neurons[END_REF], this suggests that these TLS/Sam68 clusters correspond to RNA granules (see Discussion).

Dual immunolabelling also indicated that some of the TLS clusters colocalized with NR1 clusters (Fig. 2L-N, arrows), suggesting that TLS may locate close to glutamatergic synapses.

To examine more closely the intra-dendritic localization of TLS, we used mature (4 week-old) cultures of hippocampal neurones. The dendritic accumulation of TLS was confirmed in triply stained neurones such as that of of Fig. 3A-D, in which TLS was labelled together with MAP2 (as a marker for dendritic trunks) and phalloidin (to highlight the F-actin-rich dendritic spines). Higher magnification view of the dendrites again showed that TLS condensed in inserm-00451275, version 1 -28 Jan 2010 granules in the midst of diffuse staining (Fig. 3E). Of note, TLS granules were repeatedly found at the tip of dendritic spines (arrows in Fig. 3E andF), clearly outside of the dendritic trunk. These results were again confirmed by confocal microscopy. TLS was largely absent from the nucleus (Fig. 3, I-K). Instead, TLS clusters were found in the cell body and along the entire length of the dendrites (Fig. 3, L-N).

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Discussion

The recent finding that TLS copurifies with the NMDA receptor complex [9] raises the intriguing possibility that both proteins may interact at the synapse. To our surprise, TLS was largely excluded from the nuclei of hippocampal neurones, in adult hippocampus in situ as well as in mature neuronal cultures. This suggests massive nuclear export of the protein.

Alternatively, it is possible that TLS undergoes localized degradation in the nucleus. Indeed, compared to its abundance in other tissues, TLS is relatively scarce in the brain [START_REF] Melot | Characterization of a new brain-specific isoform of the EWS oncoprotein[END_REF].

Whatever its mechanism, the depletion of nuclear TLS may conceivably play a role in neurone-specific transcription or splicing events. In cultured cell lines, increasing the intranuclear concentration of TLS has been shown either to inhibit or to potentiate the activity of specific transcription factors, and to bias exon selection in transcripts encoded by a transfected minigene [START_REF] Hallier | The Transcription Factor Spi-1/PU.1 Interacts with the Potential Splicing Factor TLS[END_REF]. Whether the lack of nuclear TLS may affect the sensitivity of neurones to DNA breaks, e.g. by ionizing radiation [START_REF] Hicks | Fus deficiency in mice results in defective B-lymphocyte development and activation, high levels of chromosomal instability and perinatal death[END_REF], is an open question.

Biochemical analysis indicated that in the cytoplasm, TLS was partitioned into a cytosolic pool and a particulate pool, sedimenting with the polysomal fraction. Diffuse and granular pools of TLS were also observed by immunofluorescence in the somatodendritic domain. The dendritic puncta often contained Sam68, an RNA-binding protein which, outside of the nucleus, becomes incorporated in RNA-containing dendritic granules [START_REF] Ben Fredj | Depolarization-induced translocation of the RNA-binding protein Sam68 to the dendrites of hippocampal neurons[END_REF][START_REF] Grange | Somatodendritic localization and mRNA association of the splicing regulatory protein Sam68 in the hippocampus and cortex[END_REF]. These observations suggest that some of the observed TLS clusters are in fact RNA granules, involved in dendritic mRNA transport or translation. It is possible that part of the punctuate immunofluorescence in fact arose from reaction of the antibody with the 100 kD protein seen in the polysomal fraction. Whether this band represents a modified, granule-specific form of TLS, or an antigenically related protein remains to be determined. However, the presence of bona fide TLS in dendritic granules is supported by several lines of evidence. A small but significant fraction of the 75 kD TLS protein was found in the polysomal pellet. Furthermore, inserm-00451275, version 1 -28 Jan 2010 when transiently expressed in hippocampal neurones, myc-tagged TLS also clustered into dendritic puncta (unpublished data). Finally, a recent proteomic study identified TLS as one of the multiple RNA-binding proteins found in biochemically purified RNA transport granules [START_REF] Kanai | Kinesin Transports RNA: Isolation and Characterization of an RNA-Transporting Granule[END_REF]. Our data provide a morphological correlate to this biochemical result. The microscopically defined granules correspond to very large supramolecular complexes, in which mRNAs, ribosomes, motor proteins, and multiple RNA-binding proteins are associated [START_REF] Kanai | Kinesin Transports RNA: Isolation and Characterization of an RNA-Transporting Granule[END_REF]. TLS might possibly become recruited from the cytosolic TLS pool during the cytoplasmic maturation of these complexes.

In adult hippocampus, a subset of TLS puncta also colocalized with NMDA receptor clusters; furthermore, in cultured neurones, TLS puncta could be seen within spine heads. Thus, the biochemical association of TLS to NMDA receptors likely occurs in the spines. Our data suggest that TLS is in an ideal position to regulate mRNA docking or translation at excitatory post-synaptic sites, as a function of the local activity history. It will be important to determine the extent and functional effect of TLS association with mRNA in the dendrites and in the spines.

Figure legends

Fig. 1: TLS is a predominantly cytoplasmic protein in hippocampal tissue. A. Fractionation scheme (see [START_REF] Feng | Fragile X Mental Retardation Protein: Nucleocytoplasmic Shuttling and Association with Somatodendritic Ribosomes[END_REF]). Total hippocampal homogenate (Hmg) was fractionated by successive centrifugation steps to yield the indicated pellets (P) and supernatants (S). Major components of each subcellular fraction are indicated. Note that the heavy membranes (P2) include intact synaptoneurosomes and mitochondria; the polysomal pellet also contains light (internal) membranes. B. Equal protein amounts (30 µg) of each subcellular fraction were loaded onto an SDS gel and analyzed by immunoblotting with antibody against TLS (top) or Sam68 (bottom). Arrow: TLS protein. Arrowhead: 100 kD protein of unknown identity.

Fig. 2 :

 2 Fig. 2: TLS localization in the hippocampus. A, B: specificity of the TLS antibody. HEK-293

Fig. 3 :

 3 Fig.3: TLS exists in dendritic granules, some of which are localized within spines. A-H: fetal

Figure

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Acknowledgements

We thank Alexei Grichine and the Institut Albert Bonniot for access to the confocal microscope and for advice in imaging. Work funded by the INSERM and by ACI Télémédecine (image analysis).

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