Panel A shows a timeline schematic of MEF treatment with nocodazole and drug exposure from minus 4 to 2 hours, marking mitosis and early G1. Panel B shows a bar graph of normalized Malat1 B RNA levels in MEFs under asynchronous and release conditions with cycloheximide treatment. Panel C shows a bar graph of normalized MALAT1 RNA levels in retinal pigment epithelial cells under asynchronous and release conditions with cycloheximide treatment. Panel D shows a bar graph of normalized Malat1 B RNA levels in MEFs after 2 hours of release with cycloheximide or Smg1 inhibitor 11e treatment. Panel E shows fluorescence microscopy images of Malat1 and merged Malat1 with 4 prime,6-diamidino-2-phenylindole staining after 2 hours of release with no drug, cycloheximide, or Smg1 inhibitor 11e.
Translation- and Smg1-mediated decay of cytoplasmic Malat1. (A) Schematic of mitotic arrest and release experimental setup. Asynchronous cells (Asy) were treated for 4 h with 50 ng/ml Noc, followed by shake-off and release of mitotic cells in Noc-free media for 2 h in the absence of presence of drug. Cells collected by shake-off at 0 h after release represent mitotic (M) cells, while cells harvested at 2 h after release represent early G1 cells. (B and C) qRT-PCR analysis of normalized Malat1 (B) or MALAT1 (C) RNA levels in p53-deficient MEFs or RPE cells, respectively, harvested at indicated time points postmitotic arrest with 50 ng/ml Noc in the absence or presence of 50 μg/ml Chx, as described in A. (D) qRT-PCR analysis of normalized Malat1 RNA levels in p53-deficient MEFs, harvested at 2 h pmr in the presence of no drug, 50 μg/ml Chx, or 5 μM hSmg1 inhibitor 11e (11e), as described in A. (E) smRNA-FISH detection of Malat1 (red) in cells fixed at 2 h pmr in the presence of no drug, 50 μg/ml Chx, or 5 μM hSmg1 inhibitor 11e (11e), as described in A. DNA, DAPI (blue). Scale bar: 20 μM. Numbers below indicate the fraction of cells with 50 or more cytoplasmic Malat1 foci in three biological replicates, with n > 50 cells scored per replicate. In this figure, bar graphs (B–D) show individual values and mean ± SD of n ≥ 3 biological replicates. Paired t test (B–E), *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.