Panel A shows fluorescence in situ hybridization images of M Y O 19 intron 11 in wild type and D B R 1 knockout cells. The intron signal is shown in red and nuclei are stained with D A P I in blue. D B R 1 knockout cells display visibly increased R N A foci compared to wild type. Panel B presents box plots of R N A foci counts and cytoplasmic localization of R N A foci. The left plot shows increased R N A foci counts in D B R 1 knockout cells compared to wild type. The right plot shows a higher percentage of cytoplasmic foci in D B R 1 knockout cells. Individual data points are overlaid on the box plots. Panel C shows lariat P C R analysis separating cytoplasmic and nuclear fractions from wild type and D B R 1 knockout cells. The results indicate enrichment of intron lariats containing inverted repeat A l u elements in the cytoplasm of D B R 1 knockout cells. A control intron without inverted repeat A l u is shown, and X I S T serves as a control. Panel D presents a dot blot assay using RNA dilutions probed with the J 2 antibody. The blot confirms that the antibody specifically recognizes A l u double stranded R N A and R N ase 3 treated samples show reduced signal, demonstrating specificity for double stranded R N A. Panel E shows immunofluorescence images of wild type and D B R 1 knockout cells stained with the J 2 antibody in red and D A P I in blue. D B R 1 knockout cells exhibit stronger cytoplasmic double stranded R N A staining. Additional panels show treatment with D N ase, R N ase H, and R N ase R, confirming that the signal corresponds to double stranded R N A rather than D N A or single stranded R N A. Panel F provides a box plot quantifying cytoplasmic double stranded R N A signal intensity. D B R 1 knockout cells show significantly higher cytoplasmic double stranded R N A signal compared to wild type, and R N ase R treatment reduces the signal. Panel G includes a bar graph showing the mean ratio of read coverage across inverted repeat A l u in cytoplasmic total versus J 2 immunoprecipitation R N A sequencing samples, comparing wild type and D B R 1 knockout cells. Panel H shows a bar graph of intron double stranded R N A expression relative to 3 prime untranslated regions in cytoplasmic J 2 immunoprecipitation R N A sequencing samples. Expression is stratified by A l u content categories, and D B R 1 knockout cells show increased intron double stranded R N A expression, particularly for inverted repeat A l u containing introns. Together, the panels demonstrate that loss of D B R 1 leads to cytoplasmic accumulation of intron lariats containing inverted repeat A l u elements and increased double stranded R N A signal.
Lariats accumulate in the cytoplasm and form dsRNA in the absence of DBR1. (A) Imaging of FISH targeted to MYO19 intron 11 (red) in WT and DBR1 KO cells. DAPI (blue) served as a marker for nuclei. The white dotted line outlines the cell boundary. The scale bar represents 5 µm. (B) Analysis of visualized RNA foci reveals an increase in foci in DBR1 KO samples, as well as a shift to the cytoplasm. n represents the number of single cells from at least three independent replicates. (C) Lariat PCR reveals specific cytoplasmic enrichment of IR Alu–containing intron lariats in DBR1 KO cells. Nuclear RNA (XIST) is used to demonstrate efficient nuclear/cytoplasmic fractionation. Data are representative of three independent experiments. (D) Dot blot demonstrating specificity of J2 antibody for Alu dsRNA relative to RNase III–digested Alu dsRNA and Alu ssRNA. (E) J2 IF of WT (top row) and DBR1 KO cells (bottom row). Cells were stained for dsRNA (J2, red), with DAPI (blue) serving as a nuclear marker. Representative images of untreated and DNase/RNase-treated cells from at least three biological replicates are shown. The scale bar represents 10 µm. (F) Quantification of cytoplasmic dsRNA signal intensity from the IF images in E (P value from t test, **P < 0.01, ***P < 0.001). (G) Mean ratio of read coverage across IR Alu in cytoplasmic total vs. J2-IP RNA-seq samples. (H) For gene regions stratified by the indicated Alu content categories, the relative expression of introns compared with 3′ UTRs in cytoplasmic J2-IP RNA-seq samples. Source data are available for this figure: SourceData F3.