Panel A shows a schematic diagram of full-length Malat1 and deletion constructs delta A, delta B, delta C, and delta D, with primer M1 and ENE mascRNA regions indicated. Panel B shows fluorescence microscopy images of Malat1 with 4′,6-diamidino-2-phenylindole staining in knockout cells treated with doxycycline, comparing empty vector, full-length, and delta A, delta B, delta C, and delta D constructs. Panel C shows three histograms of fluorescence-activated cell sorting analysis for knockout cells expressing empty vector, full-length Malat1, or delta A, with cell count on the y-axis and PE-Texas Red-A fluorescence on the x-axis. Panel D shows bar graphs measuring Malat1 A, Malat1 B, Malat1 C, and Malat1 D RNA levels in wild-type and knockout mouse embryonic fibroblasts treated with doxycycline and rescued with empty vector, full-length, or delta A, delta B, delta C, and delta D constructs. Panel E shows a schematic diagram of full-length Malat1, delta A, and delta 9xATG constructs, alongside a bar graph showing Malat1 B RNA levels in knockout cells rescued with full-length, delta A, or delta 9xATG constructs.
Characterization of Malat1 WT and mutant complementation constructs, related to Fig. 4. (A) Schematic of Doxy-regulated FL and region A–D deletion Malat1 constructs (ΔA–ΔD), expressed downstream of a TRE cassette in a PiggyBac (PB) vector, co-expressing rtTA (PB-TRE-rtTA). All constructs contain region E, required for the formation of the stabilizing ENE. (B) smRNA-FISH detection of Malat1 (red) in Malat1 KO MEFs, complemented with empty vector (EV), FL, or ΔA–ΔD Malat1, cultured in the presence of 1 μg/ml Doxy for 3 days. DAPI, DNA (blue). Scale bar: 10 μM. Representative images show that B–D region deletions affect speckle localization, while region A deletion does not. (C) FACS analysis of indicated cells, cultured in the presence of 1 μg/ml Doxy for 3 days, shows comparable cell cycle distributions in EV, FL, and ΔA-expressing KO cells. (D) qRT-PCR analysis of normalized Malat1 levels using primers located in indicated regions A–D, in WT, KO, and KO MEFs, complemented with EV, FL, or ΔA-ΔD Malat1, cultured in the presence of 1 μg/ml Doxy for 3 days. Bar graphs show individual values and mean ± SD of n = 3 technical replicates. Unpaired t test was used to perform pairwise comparisons between ΔA–ΔD and FL samples. **P < 0.01; ***P < 0.001; ns, not significant. Data indicate that ΔA and ΔC are significantly more abundant compared with FL. (E) Left, schematic of FL, ΔA, and Δ9xATG Malat1 constructs, highlighting the location of the nine mutated sORF ATGs, including M1. Right, qRT-PCR analysis of normalized Malat1 levels in Malat1 KO cells complemented with FL, ΔA, and Δ9xATG, cultured in the presence of 1 μg/ml Doxy for 3 days. Bar graph shows individual values and mean ± SD of n = 4 biological replicates. Paired t test, **P < 0.01.