Article20 August 2010free access A long nuclear-retained non-coding RNA regulates synaptogenesis by modulating gene expression Delphine Bernard Delphine Bernard Laboratoire de Biologie Cellulaire de la Synapse, Inserm 1024/CNRS 8197, Institut de Biologie de l'Ecole Normale Supérieure, Paris, FranceCo-first authors Search for more papers by this author Kannanganattu V Prasanth Kannanganattu V Prasanth Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, Urbana, IL-61801, USACo-first authors Search for more papers by this author Vidisha Tripathi Vidisha Tripathi Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, Urbana, IL-61801, USA Search for more papers by this author Sabrina Colasse Sabrina Colasse Laboratoire de Biologie Cellulaire de la Synapse, Inserm 1024/CNRS 8197, Institut de Biologie de l'Ecole Normale Supérieure, Paris, France Search for more papers by this author Tetsuya Nakamura Tetsuya Nakamura Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA Search for more papers by this author Zhenyu Xuan Zhenyu Xuan Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA Search for more papers by this author Michael Q Zhang Michael Q Zhang Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA Search for more papers by this author Frédéric Sedel Frédéric Sedel Laboratoire de Biologie Cellulaire de la Synapse, Inserm 1024/CNRS 8197, Institut de Biologie de l'Ecole Normale Supérieure, Paris, FrancePresent adress: Fédération des Maladies du Système Nerveux, Hôpital Pitié Salpétrière, Paris, France Search for more papers by this author Laurent Jourdren Laurent Jourdren IFR36, Plate-forme Transcriptome, École Normale Supérieure, Paris, France Search for more papers by this author Fanny Coulpier Fanny Coulpier IFR36, Plate-forme Transcriptome, École Normale Supérieure, Paris, France Search for more papers by this author Antoine Triller Antoine Triller Laboratoire de Biologie Cellulaire de la Synapse, Inserm 1024/CNRS 8197, Institut de Biologie de l'Ecole Normale Supérieure, Paris, France Search for more papers by this author David L Spector Corresponding Author David L Spector Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USACo-last authors Search for more papers by this author Alain Bessis Corresponding Author Alain Bessis Laboratoire de Biologie Cellulaire de la Synapse, Inserm 1024/CNRS 8197, Institut de Biologie de l'Ecole Normale Supérieure, Paris, FranceCo-last authors Search for more papers by this author Delphine Bernard Delphine Bernard Laboratoire de Biologie Cellulaire de la Synapse, Inserm 1024/CNRS 8197, Institut de Biologie de l'Ecole Normale Supérieure, Paris, FranceCo-first authors Search for more papers by this author Kannanganattu V Prasanth Kannanganattu V Prasanth Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, Urbana, IL-61801, USACo-first authors Search for more papers by this author Vidisha Tripathi Vidisha Tripathi Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, Urbana, IL-61801, USA Search for more papers by this author Sabrina Colasse Sabrina Colasse Laboratoire de Biologie Cellulaire de la Synapse, Inserm 1024/CNRS 8197, Institut de Biologie de l'Ecole Normale Supérieure, Paris, France Search for more papers by this author Tetsuya Nakamura Tetsuya Nakamura Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA Search for more papers by this author Zhenyu Xuan Zhenyu Xuan Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA Search for more papers by this author Michael Q Zhang Michael Q Zhang Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA Search for more papers by this author Frédéric Sedel Frédéric Sedel Laboratoire de Biologie Cellulaire de la Synapse, Inserm 1024/CNRS 8197, Institut de Biologie de l'Ecole Normale Supérieure, Paris, FrancePresent adress: Fédération des Maladies du Système Nerveux, Hôpital Pitié Salpétrière, Paris, France Search for more papers by this author Laurent Jourdren Laurent Jourdren IFR36, Plate-forme Transcriptome, École Normale Supérieure, Paris, France Search for more papers by this author Fanny Coulpier Fanny Coulpier IFR36, Plate-forme Transcriptome, École Normale Supérieure, Paris, France Search for more papers by this author Antoine Triller Antoine Triller Laboratoire de Biologie Cellulaire de la Synapse, Inserm 1024/CNRS 8197, Institut de Biologie de l'Ecole Normale Supérieure, Paris, France Search for more papers by this author David L Spector Corresponding Author David L Spector Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USACo-last authors Search for more papers by this author Alain Bessis Corresponding Author Alain Bessis Laboratoire de Biologie Cellulaire de la Synapse, Inserm 1024/CNRS 8197, Institut de Biologie de l'Ecole Normale Supérieure, Paris, FranceCo-last authors Search for more papers by this author Author Information Delphine Bernard1, Kannanganattu V Prasanth2,3, Vidisha Tripathi3, Sabrina Colasse1, Tetsuya Nakamura2, Zhenyu Xuan2, Michael Q Zhang2, Frédéric Sedel1, Laurent Jourdren4, Fanny Coulpier4, Antoine Triller1, David L Spector 2 and Alain Bessis 1 1Laboratoire de Biologie Cellulaire de la Synapse, Inserm 1024/CNRS 8197, Institut de Biologie de l'Ecole Normale Supérieure, Paris, France 2Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA 3Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, Urbana, IL-61801, USA 4IFR36, Plate-forme Transcriptome, École Normale Supérieure, Paris, France *Corresponding authors: Cold Spring Harbor Laboratory, One Bungtown Road, Cold Spring Harbor, NY 11724, USA. Tel.: +1 516 367 8456; Fax: +1 516 367 8876; E-mail: [email protected] de Biologie Cellulaire de la Synapse, Inserm 1024/CNRS 8197, Institut de Biologie de l'Ecole Normale Supérieure, 46 rue d'Ulm, Paris 75005, France. Tel.: +33 1 44 32 35 43; Fax: +33 1 44 32 36 54; E-mail: [email protected] The EMBO Journal (2010)29:3082-3093https://doi.org/10.1038/emboj.2010.199 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info A growing number of long nuclear-retained non-coding RNAs (ncRNAs) have recently been described. However, few functions have been elucidated for these ncRNAs. Here, we have characterized the function of one such ncRNA, identified as metastasis-associated lung adenocarcinoma transcript 1 (Malat1). Malat1 RNA is expressed in numerous tissues and is highly abundant in neurons. It is enriched in nuclear speckles only when RNA polymerase II-dependent transcription is active. Knock-down studies revealed that Malat1 modulates the recruitment of SR family pre-mRNA-splicing factors to the transcription site of a transgene array. DNA microarray analysis in Malat1-depleted neuroblastoma cells indicates that Malat1 controls the expression of genes involved not only in nuclear processes, but also in synapse function. In cultured hippocampal neurons, knock-down of Malat1 decreases synaptic density, whereas its over-expression results in a cell-autonomous increase in synaptic density. Our results suggest that Malat1 regulates synapse formation by modulating the expression of genes involved in synapse formation and/or maintenance. Introduction A large portion of the eukaryotic genome is transcribed as non-coding RNAs (ncRNAs) of various sizes ranging from ∼20 nucleotides to ∼100 kb (reviewed in Mercer et al, 2009; Wilusz et al, 2009). Despite the increasing number of long ncRNAs (lncRNAs), very few have thus far been assigned a specific function (for review, see Mercer et al, 2009). Whether some of these ncRNAs represent transcriptional noise or are involved in important cellular functions remains a matter of debate (Mattick and Makunin, 2006). However, several observations support the assertion that lncRNAs are likely to have functions in cells (Chamberlain and Brannan, 2001; Willingham et al, 2005; Feng et al, 2006; Mancini-Dinardo et al, 2006; Martianov et al, 2007; Rinn et al, 2007; Yang and Kuroda, 2007; Hirota et al, 2008; Mariner et al, 2008; Nagano et al, 2008; Wang et al, 2008; Yu et al, 2008; Chen and Carmichael, 2009; Clemson et al, 2009; Khalil et al, 2009; Mallik and Lakhotia, 2009; Sasaki et al, 2009; Sunwoo et al, 2009). For instance, large-scale studies have shown that many lncRNAs are conserved, dynamically regulated during differentiation and exhibit tissue-specific expression patterns (Ravasi et al, 2006; Dinger et al, 2008; Mercer et al, 2008; Guttman et al, 2009). In addition, several lncRNAs have been shown to be misregulated in various diseases including cancer and neurological disorders (for reviews, see Costa, 2005; Prasanth and Spector, 2007; Taft et al, 2009; Gupta et al, 2010). In many cases, the subcellular localization of ncRNAs has been determined which has provided significant insight into their functions. For example, Xist/XIST RNA, 15–17 kb in mouse and 19 kb in human, coats the inactive X chromosome from which it is transcribed. This represents part of the mechanism by which transcriptional silencing is achieved (for reviews, see Plath et al, 2002; Heard and Disteche, 2006; Payer and Lee, 2008). Recently, MEN epsilon/beta nuclear-retained ncRNAs (also known as nuclear-enriched autosomal transcript1—NEAT1) were shown to be enriched in nuclear paraspeckles, a novel subnuclear domain that preferentially localized on the periphery of nuclear speckles or interchromatin granule clusters (IGCs). These ncRNAs have a critical function in the establishment and maintenance of paraspeckles (Chen and Carmichael, 2009; Clemson et al, 2009; Sasaki et al, 2009; Sunwoo et al, 2009). Metastasis-associated lung adenocarcinoma transcript 1 (Malat1) ncRNA was initially characterized as a long polyadenylated ncRNA that is over-expressed in various cancers (Ji et al, 2003; Lin et al, 2006). Recent analysis has shown that the primary transcription products of the Malat1 locus include a 6.7 kb nuclear-retained Malat1 ncRNA, and through processing by the tRNA biogenesis machinery, a cytoplasmic 61-nt tRNA-like ncRNA referred to as mascRNA (Wilusz et al, 2008). Phylogenetic analysis indicates that Malat1 is highly conserved among mammals, up to 90% identity between human and mouse in the last 5 kb of the RNA (data not shown). Such conservation among mammals is indicative of important, yet unknown function(s) of Malat1 ncRNA. The long Malat1 transcript has been localized to nuclear speckles in several cell lines (Hutchinson et al, 2007; Clemson et al, 2009). Nuclear speckles contain a large number of nuclear proteins that are involved in several aspects of mRNP processing, including pre-mRNA splicing and RNA transport (reviewed in Lamond and Spector, 2003). Nuclear speckles are not sites of transcription or pre-mRNA splicing, but represent storage/modification and/or assembly sites of various splicing factors from where pre-mRNA processing factors are recruited to active sites of transcription (reviewed in Lamond and Spector, 2003). A population of poly(A)+ RNA was previously reported to localize to nuclear speckles (Carter et al, 1991; Visa et al, 1993; Huang et al, 1994). Malat1 is the first example of an lncRNA that is specifically enriched in these nuclear domains. The interesting sub-localization of the abundant Malat1 transcript, as well as its restricted evolutionary conservation among mammals, prompted us to investigate the potential function of Malat1 ncRNA in the mammalian cell nucleus. Here, we provide evidence that the nuclear speckle-enriched Malat1 ncRNA modulates synapse formation in neurons by regulating the expression of genes involved in synaptogenesis. Results Malat1 localizes to nuclear speckles in a transcription-dependent manner We characterized the expression of the long Malat1 ncRNA in mouse tissues by northern analysis. The ∼6.7 kb Malat1 transcript was detected in all tissue samples examined. We detected the highest levels of Malat1 ncRNA in heart, kidney and brain and a minimum level in spleen and skeletal muscle (Figure 1A; Supplementary Figure 1). As Malat1 shows elevated levels of expression in the brain, we further characterized its expression pattern by RNA fluorescence in situ hybridization (RNA-FISH) to adult mouse brain sections. We used a probe that specifically recognized the nuclear-retained 6.7 kb Malat1 RNA. We found elevated levels of Malat1 transcripts in pyramidal neurons of the hippocampus, Purkinje cells of the cerebellum, neurons of the substantia nigra and motoneurons (Figure 1B; Supplementary Figure 2). Non-neuronal cells in brain sections showed extremely low levels of Malat1 ncRNA (Figure 1C–E; arrow and open arrowheads). Interestingly, in all tissues examined, Malat1 ncRNA RNA-FISH signal displayed a punctate nuclear distribution (Figure 1C), suggesting that Malat1 ncRNA is enriched in a nuclear sub-compartment(s) that appears similar to nuclear speckles (Hutchinson et al, 2007; Clemson et al, 2009). In wild-type mouse embryonic fibroblasts (wt-MEFs), RNA-FISH revealed that Malat1 ncRNA co-localizes with the pre-mRNA-splicing factor SF2/ASF in nuclear speckles (Spector, 2006) (Figure 1F and G). In addition, Malat1 ncRNA could also be detected diffusely in the nucleoplasm (Figure 1F). Similarly, in neurons, Malat1 co-localizes with the CC3 antigen (Figure 1H–J), which is also known to localize to nuclear speckles (Chabot et al, 1995). In contrast to that observed in MEFs, the diffuse nucleoplasmic signal was significantly less in neurons (Figure 1H). The localization of Malat1 ncRNA to nuclear speckles suggests that it may execute its function in relation to gene expression (reviewed in Lamond and Spector, 2003). Figure 1.Malat1 is a neuron-enriched nuclear-retained ncRNA that is localized to nuclear speckles. (A) Northern blot analysis of various mouse tissues indicating that Malat1 is detected as a single ∼6.7 kb band with elevated levels in heart, kidney and brain. (B) Malat1 expression is restricted to neurons in the adult mouse hippocampus. RNA-FISH shows the strongest expression in pyramidal neurons of CA1 and CA3 and in granular neurons of the dentate gyrus (dg). Weaker expression can be detected in neurons of the cortex (cx) and in hippocampal interneurons (arrows). Insets show FISH signal with a sense probe (lower) and DAPI staining (upper) in CA3 region. Scale bar, 100 μm. (C–E) Triple labelling of CA3 region showing Malat1 ncRNA by in situ hybridization (C), neuron-specific NeuN immunoreactivity (D) and DAPI labelling of cell nuclei (E). Closed arrowheads indicate cells double positive for Malat1 and NeuN staining. Open arrowheads indicate non-neuronal cells. In a few cases (arrow), weak Malat1 ncRNA signal could be detected in NeuN negative cells. Scale bar, 25 μm. (F–J) Malat1 ncRNA is enriched in nuclear speckles. In wild-type mouse embryonic fibroblasts (wt-MEFs) and in mouse cultured hippocampal neurons, Malat1 ncRNA signal (F, H) co-localizes with SF2/ASF (G) or CC3 immunoreactivity (I), respectively. (J) DAPI staining. Scale bar, 10 μm. Download figure Download PowerPoint Next, we examined the behaviour of Malat1 ncRNA upon inhibition of RNA polymerase II transcription using α-amanitin (Figure 2B and C) or 5,6-dichloro-1-β-D-ribobenzimidazole (DRB; Figure 2H–K). Both drugs efficiently inhibit RNA pol II-mediated transcription. However, α-amanitin-mediated transcription inhibition is irreversible, whereas transcription in the DRB-treated cells can be reactivated upon removal of the drug from the medium. Both treatments resulted in the re-distribution of Malat1 ncRNA from nuclear speckles to a homogenous nuclear localization. However, real-time RT–PCR showed little to no turnover of Malat1 ncRNA even after prolonged treatment of cells with α-amanitin (Figure 2C) or DRB (data not shown). As previously reported (Bubulya et al, 2004), SF2/ASF localized around the nucleoli in RNA pol II transcription-inhibited cells and relocalized back to the nuclear speckles upon transcription reactivation (Supplementary Figure 3). DRB recovery kinetics revealed that within 15 min of drug removal from the culture medium, SF2/ASF could be detected within nuclear speckles, whereas Malat1 ncRNA continued to show a diffuse nuclear distribution (Figure 2L–O). However, within 30 min of recovery, Malat1 ncRNA was once again enriched in nuclear speckles in which it co-localized with SF2/ASF (Figure 2P–S). Together, these data show that Malat1 ncRNA is a very stable nuclear transcript that associates with nuclear speckles in a transcription-dependent manner. Figure 2.Malat1 ncRNA is a stable nuclear RNA that localizes to speckles in a transcription-dependent manner. (A) RNA-FISH to untreated wt-MEFs shows the nuclear speckle localization of Malat1 ncRNA. (B) α-amanitin treatment (50 μg/ml for 6 h) of wt-MEFs results in the re-distribution of Malat1 ncRNA from nuclear speckles to a homogenous nuclear distribution. (C) Malat1 ncRNA levels in untreated and α-amanitin-treated (6, 12 and 18 h) wt-MEFs were assessed by Q–PCR. Malat1 ncRNA showed little to no turnover, similar to the RNA pol III-transcribed ncRNA 7SK. As expected, the protein coding mCAT2 mRNA showed high turnover after α-amanitin treatment. Malat1, mCAT2 and 7SK RNA levels were normalized to β-actin mRNA and were presented relative to RNA levels in untreated (control) cells. The data represents mean and s.d. values of three independent experiments per data point. (D–G) RNA-FISH to wt-MEFs (untreated cells) shows complete co-localization of Malat1 ncRNA with a nuclear speckle marker SF2/ASF. (H–K) Malat1 ncRNA shows homogenous nuclear distribution upon inhibition of RNA pol II transcription by DRB (32 μg/ml for 3 h) and it no longer co-localizes with SF2/ASF. (L–O) Following the removal of DRB from the medium (15 min), Malat1 ncRNA continues to show a homogenous distribution, whereas SF2/ASF relocalizes to nuclear speckles. (P–S) Malat1 ncRNA relocalizes to nuclear speckles within 30 min post-washout of DRB from the medium. RNA-FISH is shown in red (D, H, L, P), YFP-SF2/ASF in green (E, I, M, Q) and DNA is counterstained with DAPI in blue (G, K, O, S). Scale bar, 5 μm. Download figure Download PowerPoint Malat1 modulates the recruitment of SR proteins to a transcriptionally active locus As Malat1 ncRNA is enriched in nuclear speckles, we were interested in addressing whether it has a function in regulating the recruitment of pre-mRNA-splicing factors to transcription sites. To assess such a function to a specific transcription site, we used a previously characterized U2OS cell line in which the transcription site of an inducible transgene array, as well as its mRNA and protein products, can be visualized and quantified (Janicki et al, 2004). As expected from previous experiments using this U2OS cell line (Janicki et al, 2004), the transgene locus, visualized by LacI-mCherry fluorescence, was actively decondensed upon transcriptional induction with doxycycline (compare Figure 3C and G). Upon transcriptional induction of the reporter locus in control cells that were transfected with scrambled oligonucleotides, SF2/ASF was recruited to the actively transcribing gene locus as shown by the increased number of cells displaying SF2/ASF co-localization at the transcription site (Figure 3A–H and Q). Interestingly, only ∼40–45% of the Malat1-depleted cells continued to show a speckled distribution of SF2/ASF. In the rest of the cells, SF2/ASF displayed increased homogenous nuclear distribution (data not shown). Strikingly, in a significant population of Malat1-depleted cells that continue to show speckle localization of SF2/ASF, SF2/ASF recruitment to the induced transcription site was severely compromised (Figure 3I–Q). Importantly, this was not due to a general disruption of the recruitment of the gene expression machinery as the recruitment to the activated transcription site of the rtTa transcriptional activator, CDK9, a component of the pTEF-B kinase complex that phosphorylates the C-terminal domain of RNA pol II, and RNA pol II large subunit was unaffected by knock-down of Malat1 ncRNA (Supplementary Figure 4). To determine whether Malat1 was involved in the recruitment of other splicing factors to the induced transgene, we analysed the recruitment of SC35, another member of the SR family of splicing factors. Similar to that observed for SF2/ASF, the recruitment of SC35 was also affected by the knock-down of Malat1 (Supplementary Figure 5). Figure 3.Malat1 ncRNA facilitates the recruitment of SF2/ASF to an active transcription site. (A–D) RNA-FISH to U2OS 2-6-3 cells (Janicki et al, 2004) stably expressing LacI-mCherry and rtTa transactivator (Tet-ON) shows a punctate nuclear localization of Malat1 ncRNA. Note the more homogenous nuclear staining pattern of Malat1 ncRNA in U2OS cells (A, E). In several of cancer cell lines examined, a population of cells (20–25%) showed a nuclear speckle pattern of Malat1 ncRNA unlike primary diploid cell lines and tissues where >70% of the cells exhibited a speckled distribution of Malat1 ncRNA. Cells treated with a scrambled oligonucleotide (A–D) or Malat1-specific oligonucleotide (I–L) in the absence of doxycycline (0 h DOX) do not show SF2/ASF (B, J) at the transcriptionally inactive reporter gene locus (C, K, D, L). (E–H) Upon addition of doxycycline (3 h DOX), the transcriptionally active locus (G) showed enrichment of SF2/ASF (F, H). (I–P) Cells treated with Malat1-specific antisense oligonucleotides showed complete depletion of Malat1 ncRNA (I, M). In the absence of Malat1 ncRNA, upon addition of doxycycline (3 h DOX), a significantly reduced level of SF2/ASF (N, P) was associated with the transcriptionally active locus (O). The inset in figures (C, G, K, O) represents the magnified reporter locus. Scale bar, 5 μm. (Q) The histogram shows the percentage of cells that exhibit recruitment of SF2/ASF to the transcriptionally inactive or active reporter locus in the presence or absence of Malat1 ncRNA. The data represents mean and s.d. values of three independent experiments per data point (n=25 cells/experiment). Download figure Download PowerPoint Furthermore, Malat1 knock-down did not alter the transcriptional activity of the locus as observed by RT–PCR using primers against the reporter RNA (Supplementary Figure 6). These results show that Malat1 ncRNA modulates the recruitment of SR-type pre-mRNA-splicing factors to/at active transcription sites. Importantly, knock-down of the 6.7 kb Malat1 transcript does not affect the level of mascRNA (Wilusz et al, 2008), indicating that the effect we observed is specifically due to the depletion of the long nuclear-retained Malat1 ncRNA. Malat1 ncRNA controls the mRNA levels of genes involved in the induction/function of synapses As Malat1 ncRNA is highly expressed in the brain, we investigated a possible function of Malat1 in neuronal function. We first characterized the cellular processes and components that were most impacted by Malat1 deficiency using DNA microarrays. Malat1 ASO or scrambled oligodeoxynucleotides were transfected into Neuro2A neuroblastoma cells and mRNAs were subsequently hybridized to 44K agilent DNA arrays (G_4122F). We then identified the Gene Ontology (GO) groups that were significantly enriched in the population of genes whose expression were impacted upon Malat1 depletion (Tables I and II; Supplementary Tables I, II and III). The GO groups related to the organization and the function of the nucleus were the most significantly over-represented in Malat1-depleted cells. Interestingly, we also found that the GO groups related to synapse (cellular component; GO:0045202; 1.9-fold enrichment; P=1.40 × 10−2) and dendrite development (biological process; GO: 0016358; 4.3-fold; P=6 × 10−3; see Table I) were also affected upon Malat1 depletion. No other neuron-specific GO group was significantly enriched (P<0.05). Our DNA microarray analysis shows that in neuronal cells, Malat1 preferentially regulates a subset of genes with a significant involvement in dendritic and synapse development. Table 1. Gene Ontology (GO) analysis of genes down-regulated in Malat1 knock-down Neuro2A cells GO ID Category Observed Expected Ratio P (hypergeometric) Biological processes Developmental process Dendrite development 0016358 54 5 1.17 4.29 0.006 Cellular process Regulation of cell differentiation 0045595 438 19 9.46 2.01 0.0032 Cell division 0051301 262 15 5.66 2.65 0.0006 Cell proliferation 0008283 819 31 17.68 1.75 0.0017 Mitotic cell cycle 0000278 339 17 7.32 2.32 0.0011 Mitosis 0007067 220 12 4.75 2.53 0.003 Chromosome organization 0051276 445 36 9.61 3.75 7.74e−12 Chromatine organization 0006325 347 31 7.49 4.14 1.98e−11 Nucleosome assembly 0006334 106 16 2.29 6.99 9.63e−10 Localization mRNA transport 0051028 58 5 1.25 3.99 0.037 Metabolic process Nucleobase, nucleoside, nucleotide and nucleic acid metabolic process 0006139 77 6 1.66 3.61 0.0063 Transcription 0006350 2305 82 49.76 1.65 1.57e−6 Transcription, DNA dependent 0006351 2107 74 45.49 1.63 9.79e−05 Regulation of transcription 0045449 2230 78 48.14 1.62 5.96e−06 Transcription from RNA polymerase II promoter 0006366 655 25 14.14 1.77 0.0042 DNA metabolic process 0006259 493 27 10.64 2.54 9.05e−06 DNA replication 0006260 176 18 3.8 4.74 4.84e−08 RNA metabolic process 0016070 2953 101 63.75 1.58 3.52e−07 mRNA processing 0006397 259 14 5.59 2.5 0.0015 Cellular response to stimulus 0051716 914 32 19.73 1.62 0.0047 Response to glucocorticoid stimulus 0051384 88 7 1.9 3.68 0.0029 Cellular component Synapse 0045202 373 15 7.94 1.89 0.014 Organelle 0044427 373 34 7.94 4.28 7.89e−13 Chromosome 0005694 441 35 9.38 3.73 1.89e−11 Chromatin 0000785 220 25 4.68 5.34 8.37e−12 Nucleus 0005634 4331 177 92.14 1.92 1.24e−23 Nuclear body 0016604 145 12 3.08 3.89 6.32e−05 Nucleolus 0005730 361 15 7.68 1.95 0.0107 Microtubule cytoskeleton 0015630 476 20 10.13 1.97 0.0031 The Gene Ontology groups are indicated for the biological processes and the cellular components. Category, number of reference genes in the category; Observed, number of genes in the category that are down-regulated in Neuro2A cells upon Malat1 knock-down; Expected, expected number in the category; Ratio, ratio of enrichment (R), P-value from hypergeometric test. Table 2. Gene Ontology (GO) analysis of genes up-regulated in Malat1 knock-down Neuro2A cells GO ID Category Observed Expected Ratio P (hypergeometric) Biological processes Cellular process Mitosis 0007067 220 17 6.29 2.70 0.0002 Nuclear transport 0051169 248 22 7.09 3.10 2.66e−06 Cytoskeleton organization 0007010 427 23 12.21 1.88 0.0028 Microtubule-based movement 0007018 104 10 2.97 3.36 0.0008 Metabolic process Lipid metabolic process 0006629 782 35 22.36 3.68 0.0058 Glucose metabolic process 0006006 139 10 3.97 2.52 0.0067 Ubiquitin-dependent protein catabolic process 0006511 165 15 4.72 3.18 7.87e−05 Protein folding 0006457 132 12 3.77 3.18 0.0004 Translation 0006412 436 24 12.47 1.92 0.0017 Cellular component Organelle Endoplasmic reticulum membrane 0005789 491 24 13.60 1.76 0.0053 Perinuclear region of cytoplasm 0048471 241 15 6.68 2.25 0.003 Mitochondrion 0005739 1018 40 28.20 1.42 0.016 Endosome 0005768 270 15 7.48 2.01 0.0085 Golgi apparatus 0005794 759 33 21.03 1.57 0.0071 Ribosome 0005840 236 14 6.54 2.14 0.0062 Vesicle 0031982 593 28 16.43 1.7 0.0043 Vacuole 0005773 217 16 6.01 2.66 0.0004 Nucleus 0005634 4331 148 119 1.23 0.0015 Nucleolus 0005730 361 19 10 2.14 0.0058 Cytoskeleton Spindle 0005819 99 10 2.74 6.02 0.0004 Microtubule 0005874 234 17 6.48 2.62 0.0003 Centrosome 0005813 176 11 4.88 2.26 0.01 The Gene Ontology groups are indicated for the biological processes and the cellular components. Category, number of reference genes in the category; Observed, number of genes in the category that are up-regulated in Neuro2A upon Malat1 knock-down; Expected, expected number in the category; Ratio, ratio of enrichment (R), P-value from hypergeometric test. Malat1 RNA levels in cultured neurons influence synapse formation On the basis of the DNA microarray analysis, we further investigated whether Malat1 might be involved in the generation of synapses. We first examined the developmental expression of Malat1 ncRNA. In the hippocampus as well as in Purkinje cells (data not shown), Malat1 is first detected between post-natal day 0 (P0) and P7 and its level increases until P28 (Figure 4A–E). In rodent brain, the first post-natal weeks are known to be periods of intense synaptogenesis on dendrites (Figure 4F–J) (Steward and Falk, 1986, 1991; De Felipe et al, 1997). Figure 4.Malat1 regulates genes involved in synaptogenesis in cultured hippocampal neurons. (A–J) Developmental time course of Malat1 RNA-FISH signal (A–E) and synapsin I i
