Fig 2: Distinct ribonucleoprotein complexes are formed with nucleolar-associated EU-RNA and HSATII RNA aggregates.(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 m5C-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. Blot shows singletons, but representative of experimental triplicates. -D+E, −DOX+EU samples; +D+E, +DOX+EU samples; +D-E, +DOX-EU samples. (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. N ≥ 35 ROI. ROI within the nucleoplasm is drawn with relatively the same circumference as EU-RNA foci ROI. Data are representative of two independent experimental replicates. 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. N ≥ 35 ROI. ROI within the nucleoplasm is drawn with relatively the same circumference as HSATII RNA foci ROI. Data are representative of two independent experimental replicates. 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. N ≥ 30 ROI. ROI within the nucleoplasm is drawn with relatively the same circumference as EU-RNA foci ROI. Data are representative of two independent experimental replicates. 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. N ≥ 90 ROI. ROI within the nucleoplasm is drawn with relatively the same circumference as HSATII RNA foci ROI. Data are representative of three independent experimental replicates. 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 ≥ 200 nuclei. Dots represent averages of three experimental replicates graphed. Data represent means ± SD. Statistical differences between groups were analyzed employing two-tailed Unpaired t-test.

Fig 3: NSUN2 activity is necessary for YBX-1 association with HSATII RNA.(A) Combined immunofluorescence and HSATII RNA-FISH of HSATII (green) and YBX-1 (magenta) in +DOX iDUX4 cells either pre-treated with DMSO or 5μM 5-azaC for 24-hours, then dox-induced and fixed 24 hours post-induction. Scale bar = 10μm. Images are representative of three independent experimental replicates performed. (B) Percent of HSATII+ nuclei with YBX-1 signal that overlap with HSATII RNA foci in +DOX iDUX4 cells with or without 5-azaC treatment. N ≥ 200 nuclei. Dots represent mean of independent experimental replicates. Data represent means ± SD. Statistical differences between groups were analyzed employing two-tailed Unpaired t-test. (C) Combined immunofluorescence and HSATII RNA-FISH of HSATII (green) and m5C (magenta) in +DOX iDUX4 cells either pre-treated with siRNAs targeting control sequences (siCTRL) or NSUN2 (siNSUN2) for 24-hours, then dox-induced and fixed 24 hours post-induction. Scale bar = 20μm. Images are representative of three independent experimental replicates performed. (D) Mean signal intensity of m5C within specified ROI in the nucleus: either within HSATII RNA foci or the remainder of the nucleoplasm in +DOX HSATII+ iDUX4 cells with control (siCTRL) or NSUN2 (siNSUN2) depletion. Dots are each individual ROI. N ≥ 50 ROI. ROI within the nucleoplasm is drawn with relatively the same circumference as HSATII RNA foci ROI. Data are representative of three independent experimental replicates. Data represent means. Statistical differences between groups were analyzed employing one-way ANOVA Tukey’s multiple comparison test. (E) Combined immunofluorescence and HSATII RNA-FISH of HSATII (green) and YBX-1 (magenta) in +DOX iDUX4 cells either pre-treated with siRNAs targeting control sequences (siCTRL) or NSUN2 (siNSUN2) for 24-hours, then dox-induced and fixed 24 hours post-induction. Scale bar = 20μm. Images are representative of three independent experimental replicates performed. (F) Percent of HSATII+ nuclei with YBX-1 signal that overlap with HSATII RNA foci in +DOX iDUX4 cells with control (siCTRL) or NSUN2 (siNSUN2) depletion. N ≥ 200 nuclei. Dots represent fields taken from representative experiment. Data represent means ± SD. Statistical differences between groups were analyzed employing Mann Whitney test. (G) Percent of nuclei with YBX-1 nuclear aggregates in +DOX iDUX4 cells with control (siCTRL) or NSUN2 (siNSUN2) depletion. N ≥ 200 nuclei. Dots represent fields taken from representative experiment. Data represent means ± SD. Data are representative of three independent experimental replicates. Statistical differences between groups were analyzed employing Mann Whitney test.

Fig 4: Analysis of m5C RNA methylation regulators. (A) Analysis of the relative expression of eight RNA methylation regulators in BC and adjacent tissues by qPCR. The expression of NSUN2 in BC and BCBM was quantified by qPCR (B) and western-blot (C-D, Supplementary Original western blots). NSUN2 knockdown efficiency in BCBM cells measured by immunofluorescent (E, scale bars: 100 μm), qPCR (F), and Western-blot (G-H, Original blots/gels are shown in Supplementary Original western blots). (I) The relative expression levels of 7 different methylation genes in BCBM following NSUN2 knock-down. (n = 3; Data are presented as the mean ± SD; P < 0.05)