Panel A: The microscopy image shows fluorescence images of cells stained for Malat1 (red), Srrm2 (green), and DNA (blue). The illustration depicts the distribution of Malat1 in different cell cycle stages. Panel B: The fluorescence images of cells stained for Malat1 (green), Neat1-5 (red), and DNA (blue) in early G1 and G1/S/G2 phases. Panel C: A schematic diagram illustrating the synchronization strategy of cells and showing cell cycle stage distribution at different time points. Panel D: One immunoblot image showing Cyclin B1 levels at various time points post mitotic release. Panel E: One bar graph showing the relative abundance of Rn7s1, Kcnq1ot1, and Malat1 in nuclear and cytosolic fractions at different time points. Panel F: One bar graph showing relative RNA levels of Malat1 detected with different primer sets at various time points. Panel G: Two line graphs showing normalized Malat1 levels in cells treated with Actinomycin D and 5-ethynyl uridine over time.
Degradation of mitotically inherited cytoplasmic Malat1 in early G1. (A) Top, representative images of smRNA-FISH/IF detection of Malat1 (red) and nuclear speckle marker, Srrm2 (green), in WT1 primary MEFs at passage 4. DAPI, DNA (blue). Scale bar: 10 μM. Bottom, schematic of representative Malat1/DAPI patterns in mitosis (M), early G1, or G1/S/G2 cell cycle stages. (B) smRNA-FISH co-detection of Malat1 (green) and Neat1-5′ (red) in cells from A with presence (early G1) or absence (G1/S/G2) of cytoplasmic Malat1 staining. DNA, DAPI (blue). Arrowheads indicate co-localization of Malat1 and Neat1-5′ signals at sites of transcription in early G1. Scale bar: 10 μM. (C) Top, schematic of mitosis synchronization strategy of p53-deficient MEFs by treatment with 50 ng/ml Noc for 4 h, followed by manual shake-off of mitotic cells, and release into Noc-free media for the indicated time. Bottom, cell cycle stage distribution by FACS of cells collected at indicated time points post mitotic release (pmr). (D) Immunoblot detection of mitotic marker cyclin B1 in whole-cell extract isolated from cells, collected at indicated time points pmr, as described in C. Hsp90, loading control. Molecular weight markers, kDa. (E) qRT-PCR analysis of the relative abundance of Rn7s1 (cytosolic marker), Kcnq1ot1 (nuclear marker), and Malat1 in nuclear and cytosolic fractions from p53-deficient MEFs, harvested asynchronously (A) or at indicated time points pmr, as described in C. Bar graphs show the mean of n = 3 biological replicates. Paired t test, *P < 0.05; ***P < 0.001. (F) qRT-PCR detection of total and unprocessed relative Malat1 RNA levels, detected with primer sets specific to regions B and E, relative to Gapdh in p53-deficient MEFs, collected at indicated time points pmr, as described in C. Bar graph shows mean ± SD of n = 3 technical replicates. Paired t test, ***P < 0.001. Data were confirmed in a biological replicate. (G) qRT-PCR detection of normalized Malat1 levels in left, p53-deficient MEFs treated with 5 μg/ml ActD for the indicated time. Half-life could not be determined due to lack of observable decay over the experimental time course; and right, p53-deficient MEFs pulsed for 1 h with 0.5 mM 5-EU and released in EU-free media for the indicated time. Half-life was determined using nonlinear regression of one-phase decay. Graph shows mean ± SD of n = 3 technical replicates. Source data are available for this figure: SourceData F2.