Journal: The Journal of cell biology

Article Title: DUX4-induced HSATII RNA accumulation drives protein aggregation impacting RNA processing pathways

doi: 10.1083/jcb.202501129

Figure Lengend Snippet: ( A ) Immunofluorescence of EU-RNA aggregates (gray) in iDUX4 cells that were left untreated (−DOX) or pulsed for 4-hours using 1ug/mL doxycycline (+DOX) and then incubated with 0.1mM 5-ethynyluridine (+EU) for 16-hours prior to washout and fixed at 24-hour time points (24–96 hours). Control cells were uninduced (−DOX) iDUX4 cells incubated with EU and fixed at either 24 hours or 96 hours or without EU and fixed at 24 hours. Scale bar = 20μm. Images are representative of two independent experimental replicates performed. ( B ) Percent of cells with intranuclear EU-RNA aggregates in −DOX or +DOX iDUX4 cells labeled with EU for 16-hours at 24-hour time points. EU-RNA aggregates are present within +DOX iDUX4 cells from 24-hour to 72-hour time points. N=2 experimental replicates. Minimum 200 nuclei per experiment. Data represent means ± standard deviation (SD). Statistical differences between groups were analyzed employing one-way ANOVA Dunnett’s multiple comparisons test between each group and a control (−DOX+EU 24-hour time point). ( C ) Enrichment of RPL27 or HSATII RNA in isolated EU-RNA from −DOX or +DOX iDUX4 cells incubated with EU for 16-hours or +DOX iDUX4 cells with no EU treatment, all harvested at the 48-hour time point. Housekeeping gene RPL27 is used as a control. N=3. A total of four independent experimental replicates were performed. Data represent means ± SD. ( D ) Quantitative RT-PCR of HSATII expression (2 −DCt ) in −DOX or +DOX iDUX4 cells treated with EU for 16-hours or +DOX iDUX4 cells with no EU treatment, all harvested at the 48-hour time point. EU-treatment does not impact HSATII RNA expression. N=3. A total of four independent experimental replicates were performed. Data represent means ± SD. Statistical differences between groups were analyzed employing one-way ANOVA Dunnett’s multiple comparisons test between each group and a control (−DOX+EU). ( E ) Enrichment of RPL27 or HSATII RNA in isolated EU-RNA from −DOX or +DOX iDUX4 cells incubated with EU for 16-hours and harvested at 24-hour time points from 48-hours to 96-hours. Housekeeping gene RPL27 is used as a control. N=3. A total of three independent experimental replicates were performed. Data represent means ± SD. ( F ) Combined immunofluorescence and HSATII RNA-fluorescence in situ hybridization (RNA-FISH) of HSATII RNA (green) and EU-RNA (magenta) in +DOX+EU iDUX4 cells fixed at 48-hour time point. Scale bar = 20μm. Images are representative of three independent experimental replicates performed. ( G ) Percent of cells with intranuclear EU-RNA aggregates only (“EU”), HSATII RNA aggregates only (“HSATII”) or both EU-RNA/ HSATII RNA aggregates (“both”) in +DOX+EU iDUX4 cells fixed at the 48-hour time point. N=3 experimental replicates. Minimum 200 nuclei per experiment. Data represent means ± SD. ( H ) Combined immunofluorescence and HSATII RNA-FISH of HSATII RNA (green) and EU-RNA (magenta) in control depleted (“CTRL KD”) or HSATII RNA-depleted (“HSATII KD”) +DOX+EU iDUX4 cells fixed at 48-hour time point. Scale bar = 20μm. Images are representative of two independent experimental replicates performed. ( I ) Percent of cells with intranuclear RNA aggregates: EU only, HSATII only or both in CTRL KD or HSATII KD +DOX+EU iDUX4 cells fixed at the 48-hour time point. N=2 experimental replicates. Minimum 200 nuclei per experiment. Data represent means ± SD. Statistical differences between groups were analyzed employing two-way ANOVA Šídák’s multiple comparisons test between CTRL KD and HSATII KD within each group (EU, HSATII, both). Fig 1G and 1L : The frequency of cells containing RNA aggregates exhibits some variability across the population. This variability could be due to the robustness and induction efficiency of doxycycline treatment. ( J ) Combined immunofluorescence (IF) of EU-RNA (green) and Fibrillarin (magenta) in −DOX+EU or +DOX+EU iDUX4 cells fixed at 48-hour time point or combined immunofluorescence and HSATII RNA-FISH of HSATII RNA (green) and fibrillarin (magenta) in −DOX and +DOX iDUX4 cells fixed at 24-hours. Scale bar = 20μm. Arrows indicate nuclei with disrupted nucleolar staining. Images are representative of two independent experimental replicates performed. ( K ) Immunofluorescence (IF) of EU-RNA (green) and nucleophosmin-1 (NPM1) (magenta) in −DOX+EU or +DOX+EU iDUX4 cells fixed at 48-hour time point or combined immunofluorescence and HSATII RNA-FISH of HSATII RNA (green) and NPM1 (magenta) in −DOX and +DOX iDUX4 cells fixed at 24-hours. Scale bar = 20μm. Arrows indicate nuclei with disrupted nucleolar staining. Images are representative of two independent experimental replicates performed. ( L ) Percent of cells with nucleolar disruption present in cells with intranuclear RNA aggregates. N=2 experimental replicates. Minimum 300 nuclei per experiment.

Article Snippet: RNA was isolated in RNA elution buffer (10 mM EDTA, pH 8.0, 95% formamide (ThermoFisher Scientific)) and incubated at 95°C for 5 minutes at 1,000xg.

Techniques: Immunofluorescence, Incubation, Control, Labeling, Standard Deviation, Isolation, Quantitative RT-PCR, Expressing, RNA Expression, Fluorescence, In Situ Hybridization, Staining, Disruption

Journal: The Journal of cell biology

Article Title: DUX4-induced HSATII RNA accumulation drives protein aggregation impacting RNA processing pathways

doi: 10.1083/jcb.202501129

Figure Lengend Snippet: ( A ) Biological process pathway analysis of DUX4-induced stable EU-RNA associated RNPs (EU-RNPs) proteomics filtered protein hits (>2 unique peptide matches and >1.5 difference) ran through Enrichr. EU-RNP complexes were isolated from −DOX+EU, +DOX+EU or +DOX-EU iDUX4 cells harvested at the 48-hour time point using the “RICK” approach ( Bao et al., 2018 ). Protein was purified from isolated EU-RNP complexes and was subjected to mass-spectrometry. Proteomics was performed in experimental duplicates. ( B ) Validation of selected proteins identified through EU-RNP proteomics: Known DUX4-induced protein aggregates SC35 and TDP-43, and m 5 C-related factors NSUN2 and YBX-1. Proteins were isolated as stated in Fig. 2A . GAPDH and beta-tubulin were used as loading controls for inputs and negative controls for pulldown samples. 5% input was removed prior to RNA pulldown. N=1 per condition. A total of three independent experimental replicates were performed. −D+E, −DOX+EU samples; +D+E, +DOX+EU samples; +D-E, +DOX-EU samples. Quantification of immunoblot is shown right. ( C ) Immunofluorescence of EU-RNA (green) and SC35 (magenta) in −DOX+EU or +DOX+EU iDUX4 cells fixed at 48-hour time point. Scale bar = 20μm. Images are representative of two independent experimental replicates performed. ( D ) Mean signal intensity of SC35 within specified regions of interest (ROI) in the nucleus: either within EU-RNA foci or the remainder of the nucleoplasm in +DOX EU-RNA+ nuclei in iDUX4 cells. Dots are each individual ROI. ROI within the nucleoplasm is drawn with relatively the same circumference as EU-RNA foci ROI. N= 50 ROI.. A total of two independent experimental replicates were performed. Data represent means. Statistical differences between groups were analyzed employing Mann Whitney test. ( E ) Combined immunofluorescence and HSATII RNA-FISH of HSATII (green) and SC35 (magenta) in −DOX or +DOX iDUX4 cells fixed at 24-hour time point. Scale bar = 20μm. Images are representative of two independent experimental replicates performed. ( F ) Mean signal intensity of SC35 within specified ROI in the nucleus: either within HSATII RNA foci or the remainder of the nucleoplasm in +DOX HSATII+ nuclei in iDUX4 cells. Dots are each individual ROI. ROI within the nucleoplasm is drawn with relatively the same circumference as HSATII RNA foci ROI. N= 35 ROI.. A total of two independent experimental replicates were performed. Data represent means. ( G ) Immunofluorescence of EU-RNA (green) and TDP-43 (magenta) in −DOX+EU or +DOX+EU iDUX4 cells fixed at 48-hour time point. Scale bar = 20μm. Images are representative of two independent experimental replicates performed. ( H ) Mean signal intensity of TDP-43 within specified ROI in the nucleus: either within EU-RNA foci or the remainder of the nucleoplasm in +DOX EU-RNA+ nuclei in iDUX4 cells. Dots are each individual ROI. ROI within the nucleoplasm is drawn with relatively the same circumference as EU-RNA foci ROI. N= 30 ROI.. A total of two independent experimental replicates were performed. Data represent means. Statistical differences between groups were analyzed employing Mann Whitney test. ( I ) Combined immunofluorescence and HSATII RNA-FISH of HSATII (green) and TDP-43 (magenta) in −DOX or +DOX iDUX4 cells with CTRL KD or HSATII KD fixed at 24-hour time point. Scale bar = 20μm. Images are representative of three independent experimental replicates performed. ( J ) Mean signal intensity of TDP-43 within specified ROI in the nucleus: either within HSATII RNA foci or the remainder of the nucleoplasm in +DOX HSATII+ nuclei in iDUX4 cells. Dots are each individual ROI. ROI within the nucleoplasm is drawn with relatively the same circumference as HSATII RNA foci ROI. N= 90 ROI. A total of three independent experimental replicates were performed. Data represent means. Statistical differences between groups were analyzed employing Mann Whitney test. ( K ) Percent of cells with TDP-43 nuclear aggregates in +DOX iDUX4 cells with CTRL KD or HSATII KD. N=3 experimental replicates. Minimum 200 nuclei per experiment. Data represent means ± SD. Statistical differences between groups were analyzed employing two-tailed Unpaired t-test.

Article Snippet: RNA was isolated in RNA elution buffer (10 mM EDTA, pH 8.0, 95% formamide (ThermoFisher Scientific)) and incubated at 95°C for 5 minutes at 1,000xg.

Techniques: Isolation, Purification, Mass Spectrometry, Biomarker Discovery, Western Blot, Immunofluorescence, MANN-WHITNEY, Two Tailed Test

Journal: The Journal of cell biology

Article Title: DUX4-induced HSATII RNA accumulation drives protein aggregation impacting RNA processing pathways

doi: 10.1083/jcb.202501129

Figure Lengend Snippet: ( A ) Experimental schematic showing ChIRP protocol ( Chu et al., 2012 ). ( B ) RT-qPCR showing enrichment of RPL27 , ZSCAN4 or HSATII RNA in ChIRP pulldowns using either control ASOs or HSATII-specific ASOs in +DOX iDUX4 cells. N=3 biological replicates per experiment (small black points). A total of three independent experimental replicates were performed (colored points). ( C ) Immunoblot showing enrichment of known HSATII interacting proteins MeCP2 and eIF4A3 in ChIRP pulldowns (PD) using either control ASOs (CTRL) or HSATII-specific ASOs (HSATII) in −DOX or +DOX iDUX4 cells. GAPDH used as loading control for inputs and negative control for PD samples. 5% input was removed prior to RNA pulldown. Immunoblot shows two biological replicates. N=2 per condition. A total of three experimental replicates were performed. Quantification of immunoblot is shown below. ( D ) Biological process pathway analysis of HSATII RNA associated RNPs (HSATII-RNP) proteomics filtered protein hits (>2 unique peptide matches and >1.5 difference) ran through Enrichr. HSATII-RNP complexes were isolated from −DOX or +DOX iDUX4 cells harvested at the 24-hour time point using the “ChIRP” approach ( Chu et al., 2012 ). Protein was purified from isolated HSATII-RNP complexes using antisense oligonucleotides (ASO) targeting HSATII or control sequences and was subjected to mass-spectrometry. Proteomics was performed in biological triplicate. ( E ) Validation of selected proteins identified through HSATII-RNP proteomics: m 5 C-related factors YBX-1 and m 6 A-related factors YTHDC1, WTAP, and VIRMA. Proteins were isolated as stated in Fig. 3B . GAPDH was used as a loading control for inputs and negative control for pulldown samples. 5% input was removed prior to RNA pulldown. N=1 per condition. A total of three independent experimental replicates were performed.. CTRL, Control ASO; HSATII, HSATII-specific ASO; PD, pulldown. Quantification of immunoblot is shown right.

Article Snippet: RNA was isolated in RNA elution buffer (10 mM EDTA, pH 8.0, 95% formamide (ThermoFisher Scientific)) and incubated at 95°C for 5 minutes at 1,000xg.

Techniques: Quantitative RT-PCR, Control, Western Blot, Negative Control, Isolation, Purification, Mass Spectrometry, Biomarker Discovery

Journal: The Journal of cell biology

Article Title: DUX4-induced HSATII RNA accumulation drives protein aggregation impacting RNA processing pathways

doi: 10.1083/jcb.202501129

Figure Lengend Snippet: ( A ) Combined immunofluorescence and HSATII RNA-FISH of HSATII (green) and VIRMA (magenta) in −DOX or +DOX iDUX4 cells with CTRL KD or HSATII KD fixed at 24-hour time point. Scale bar = 20μm. Images are representative of three independent experimental replicates performed. ( B ) Percent of nuclei with VIRMA signal that at least have partial signal overlap with HSATII RNA foci in +DOX HSATII+ nuclei in iDUX4 cells. N=3 experimental replicates. Minimum 200 nuclei per experiment. Data represent means ± SD. ( C ) Mean signal intensity of VIRMA within specified ROI in the nucleus: either within HSATII RNA foci or the remainder of the nucleoplasm in +DOX HSATII+ nuclei in iDUX4 cells. Dots are each individual ROI. ROI within the nucleoplasm is drawn with relatively the same circumference as HSATII RNA foci ROI. N= 60 ROI. A total of two independent experimental replicates were performed. Data represent means. Statistical differences between groups were analyzed employing Mann Whitney test. ( D ) Percent of cells with VIRMA nuclear aggregates in +DOX iDUX4 cells with CTRL KD or HSATII KD. Data represent means ± SD. N=3 experimental replicates. Minimum 200 nuclei per experiment. Statistical differences between groups were analyzed employing Two-tailed t-test. ( E ) Combined immunofluorescence and HSATII RNA-FISH of HSATII (green) and WTAP (magenta) in −DOX or +DOX iDUX4 cells with CTRL KD or HSATII KD fixed at 24-hour time point. Scale bar = 20μm. Images are representative of three independent experimental replicates performed. ( F ) Percent of nuclei with WTAP signal that at least have partial signal overlap with HSATII RNA foci in +DOX HSATII+ nuclei in iDUX4 cells. N=3 experimental replicates. Minimum 200 nuclei per experiment. Data represent means ± SD. ( G ) Mean signal intensity of WTAP within specified ROI in the nucleus: either within HSATII RNA foci or the remainder of the nucleoplasm in +DOX HSATII+ nuclei in iDUX4 cells. Dots are each individual ROI. ROI within the nucleoplasm is drawn with relatively the same circumference as HSATII RNA foci ROI. N= 60 ROI. A total of three independent experimental replicates were performed. Data represent means. Statistical differences between groups were analyzed employing Mann Whitney test. ( H ) Percent of cells with WTAP nuclear aggregates in +DOX iDUX4 cells with CTRL KD or HSATII KD. Data represent means ± SD. N=3 experimental replicates. Minimum 200 nuclei per experiment. Statistical differences between groups were analyzed employing Two-tailed Unpaired t-test. ( I ) Combined immunofluorescence and HSATII RNA-FISH of HSATII (green) and YTHDC1 (magenta) in −DOX or +DOX iDUX4 cells with CTRL KD or HSATII KD fixed at 24-hour time point. Scale bar = 20μm. Images are representative of three independent experimental replicates performed. ( J ) Percent of nuclei with YTHDC1 signal that at least have partial signal overlap with HSATII RNA foci in +DOX HSATII+ nuclei in iDUX4 cells. Dots represent fields taken from representative experiment. N=3 experimental replicates. Minimum 200 nuclei per experiment. Data represent means ± SD. ( K ) Mean signal intensity of YTHDC1 within specified ROI in the nucleus: either within HSATII RNA foci or the remainder of the nucleoplasm in +DOX HSATII+ nuclei in iDUX4 cells. Dots are each individual ROI. ROI within the nucleoplasm is drawn with relatively the same circumference as HSATII RNA foci ROI. N= 25 ROI. A total of three independent experimental replicates were performed. Data represent means. Statistical differences between groups were analyzed employing Unpaired t-test. ( L ) Percent of cells with YTHDC1 nuclear aggregates in +DOX iDUX4 cells with CTRL KD or HSATII KD. Data represent means ± SD. N=3 experimental replicates. Minimum 200 nuclei per experiment. Statistical differences between groups were analyzed employing Two-tailed Unpaired t-test. ( M ) Combined immunofluorescence and HSATII RNA-FISH of HSATII (green) and m 6 A (magenta) in −DOX or +DOX iDUX4 cells fixed at 24-hour time point. Scale bar = 20μm. Images are representative of three independent experimental replicates performed. ( N ) Mean signal intensity of m 6 A within specified ROI in the nucleus: either within HSATII RNA foci or the remainder of the nucleoplasm in +DOX HSATII+ nuclei in iDUX4 cells. Dots are each individual ROI. ROI within the nucleoplasm is drawn with relatively the same circumference as HSATII RNA foci ROI. N= 100 ROI. A total of three independent experimental replicates were performed. Data represent means. Statistical differences between groups were analyzed employing Mann-Whitney test. ( O ) Mean signal intensity of nuclear m 6 A signal in −DOX or +DOX cells fixed at 24-hour time point. Dots are individual nuclei. N= 100 nuclei. A total of three independent experimental replicates were performed. Data represent means. Statistical differences between groups were analyzed employing One-way ANOVA Tukey’s multiple comparison test. ( P ) RNA dot blot of RNA isolated from uninduced (−DOX), control depleted (siCTRL), VIRMA depleted (siVIRMA) or +DOX iDUX4 cells harvested at 24-hours. 200ng of RNA was loaded. RNA was probed using anti-m 6 A antibody or total RNA was determined using Methylene Blue. Signal intensity showed an increase in m 6 A RNA levels in +DOX iDUX4 cells compared to uninduced cells. N=3.

Article Snippet: RNA was isolated in RNA elution buffer (10 mM EDTA, pH 8.0, 95% formamide (ThermoFisher Scientific)) and incubated at 95°C for 5 minutes at 1,000xg.

Techniques: Immunofluorescence, MANN-WHITNEY, Two Tailed Test, Comparison, Dot Blot, Isolation, Control