Panel A shows a schematic diagram of Malat1 primer positions and ribonuclease P processing. Panel B shows bar graph of relative RNA levels of Malat1 B and Malat1 E across passages in WT1 and WT2 cells. Panel C shows bar graphs of relative RNA levels of Malat1 B and Malat1 E under different serum concentrations and doxorubicin treatment. Panel D shows fluorescence microscopy images of Malat1 and merged Malat1 with 4 prime,6-diamidino-2-phenylindole staining at passages 4 and 10. Panel E shows violin plots of nuclear Malat1 measurements at passages 4 and 10 in WT1 and WT2 cells. Panel F shows a violin plot of nuclear Malat1 measurements across early G1/S/G2, G1, and S/G2 cell-cycle phases. Panel G shows a cell-cycle schematic and a bar graph of normalized Malat1 B RNA levels in green fluorescent protein-negative and green fluorescent protein-positive cells. Panel H shows fluorescence microscopy images and a violin plot comparing nuclear Malat1 levels in green fluorescent protein-negative and green fluorescent protein-positive cells. Panel I shows a bar graph of normalized Malat1 B RNA levels in mouse embryonic fibroblasts treated with RO-3306 and nocodazole. Panel J shows a bar graph of normalized Malat1 RNA levels in retinal pigment epithelial cells under untreated and doxorubicin-treated conditions.
Cell cycle–dependent regulation of Malat1 abundance. (A) Schematic of unprocessed and processed Malat1 transcripts, highlighting the location of primers in regions A–E and the RNase P processing event, which removes the mascRNA-containing region E to generate the ENE terminus. (B) qRT-PCR detection of total and unprocessed Malat1 RNA levels, detected with primer sets specific to regions B and E, relative to Gapdh in two independent primary WT MEF lines, WT1 and WT2, at indicated passages. (C) qRT-PCR detection of total and unprocessed Malat1 RNA levels, detected with primer sets specific to regions B and E, relative to Gapdh in left, primary WT1 grown in 10% serum or upon starvation in 0.5% serum or right, primary WT1 untreated or treated with 0.5 μM doxorubicin (Doxo) for 24 h. (D) Panoramic images of smRNA-FISH detection of Malat1 (red) in WT1 and WT2 MEFs from B at passage 4 and 10. DAPI, DNA (blue). * indicate cells in early G1 with cytoplasmic Malat1 signal. Scale bar: 20 μM. (E) Violin plot of nuclear Malat1 mean intensity (MI), measured in a.u., in WT1 and WT2 cells at indicated passages, quantified in images from D. (F) Violin plot of nuclear Malat1 MI, measured in a.u., in WT1 cells at passage 4 that are positive (early G1) or negative (G1/S/G2) for cytoplasmic Malat1 signal, quantified in images from D. (G) Left, schematic of FUCCI system; right, qRT-PCR analysis of normalized Malat1 levels in GFP-negative (GFP-, G1) and GFP-positive (GFP+, S/G2/M) FUCCI-labeled cells sorted by FACS. (H) Left, co-detection of Malat1 by smRNA-FISH (red) and GFP (green) in FUCCI-labeled cells. DAPI, DNA (blue). Right, violin plot of nuclear Malat1 MI, measured in a.u., in GFP- or GFP+ cells. Scale bar: 10 μM. (I) qRT-PCR analysis of normalized Malat1 levels in p53-deficient MEFs, treated with 8 μM RO-3306 or 50 ng/ml Noc, as indicated. (J) qRT-PCR analysis of normalized MALAT1 levels in RPE cells treated with 8 μM RO-3306 or 50 ng/ml Noc, as indicated. In this figure, bar graphs show individual values and mean ± SD of n ≥ 3 biological (G, I, and J) and technical (B and C) replicates. n indicates the number of nuclei represented in violin plots (E, F, and H). Paired t test (G, I, and J), unpaired t test (B, C, F, G, and H), and sample variance (E); *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.