PMC

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 immunoreactivity (IR) (F–J) in pyramidal neurons (py) of the mouse hippocampus (CA3) between post-natal day 0 (P0) and P28. First punctate signals of synapsin I-IR and first Malat1-FISH signals were detected in the stratum radiatum (sr) at P7 and increases until P28. Scale bar, 25 μm. (K) Synapsin immunoreactivity in cultured hippocampal neurons transfected with Malat1 antisense oligodeoxynucleotides (AS) or control scrambled oligodeoxynucleotide (Scr). Scale bar, 10 μm. Lower panels display higher magnifications of the outlined regions in the upper panels. Histogram shows the quantification of synaptic linear density in three independent experiments. Mean±s.e.m.; ^**P<0.0001, ANOVA. (L) Synapsin labelling (red) in neurons (green) transfected with a control vector (Ctl) or with Malat1 over-expressing vector (Malat1). Histogram shows the quantification of synaptic linear density in three independent experiments. Mean±s.e.m.; ^*P<0.001, ANOVA. (M) Synapsin labelling in neurons neighbouring control vector or Malat1 cDNA-transfected neurons. Histogram shows the quantification of synaptic linear density in three independent experiments. Mean±s.e.m.; P=0.49, ANOVA. (N) Quantification by quantitative RT–PCR of mRNA encoding several genes upon transfection of three independent cultures of neurons with Malat1 antisense oligonucleotides (AS1, grey bars; AS4, black bars) or with control scrambled oligodeoxynucleotide (Scr, white bars). Mean±s.e.m. relative to Actin mRNA level ^**P<0.03; ^*P<0.05 Mann–Whitney U-test.

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.

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.

Malat1 ncRNA facilitates the recruitment of SF2/ASF to an active transcription site. (A–D) RNA-FISH to U2OS 2-6-3 cells () 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).