Fig 2: NISM interacts with DHX9.(A) Depiction of nuclear lysate immunoprecipitation-mass spectrometry (IP-MS) analysis workflow and Venn diagram showing the overlap in prey proteins significantly enriched by short and long NISM-ALFA; n = 3. (B) STRING database plot showing interactions between the 49 prey proteins pulled down by both short and long NISM-ALFA. DExD/H-box RNA helicase family members are highlighted in red and prey proteins were grouped by k means clustering. (C-D) Immunoblot validation of DHX9 enrichment in NISM-ALFA IP samples (C) and NISM-ALFA enrichment in DHX9 IP samples (D). (E) Representative immunofluorescence images of NPM1 (red), DHX9 (cyan), and NISM-ALFA (green) in U2OS cells overexpressing short or long NISM-ALFA. All immunoblots and images are representative of at least two biological replicates.

Fig 3: NISM-ALFA rescues nucleolar formation and DHX9 localization in knockout cells.(A-B) Representative immunofluorescence images of NPM1 (red), DHX9 (cyan), and NISM-ALFA (green) in wild-type (WT) and NISM knockout (NISM KO) U2OS cells transfected with empty vector, short NISM-ALFA, or long NISM-ALFA for 48 h. Nuclei were counter-stained with DAPI (blue). Scale bar, 5 μm. All data are representative of at least two biological replicates. (C) Proposed model for how NISM perturbation regulates nucleolar dynamics. In WT U2OS cells, NISM interacts with DHX9 to enhance its LLPS propensity, thus allowing for proper formation of nucleoli. Synthesis and processing of rRNA, nucleolar R-loop homeostasis, and all steps of ribosome biogenesis proceed normally. When NISM is overexpressed, DHX9 nucleolar phase separation is increased, in turn causing a reduction in nucleolar R-loops, impaired rRNA synthesis, and the formation of stress caps at the outer edges of shrunken, rounded nucleoli. These effects lead to p53 stabilization, cell cycle checkpoint activation, and suppressed cell proliferation. In contrast to overexpression, NISM knockout causes disruption of the nucleolar structure, thus resulting in dispersion of nucleolar proteins and redistribution of DHX9 to the nucleoplasm. While rRNA synthesis and overall translation levels are not significantly impacted, p53 is stabilized and cell proliferation is strongly suppressed.

Fig 4: NISM-ALFA overexpression inhibits rRNA synthesis and reduces nucleolar R-loops and overall translation levels.(A) Representative immunofluorescence images of FBL (cyan), NISM-ALFA (green), and 5-EU (red) in wild-type (WT) U2OS cells treated with 10 nM ActD or DMSO for 3 h and transfected with empty vector (EV), short NISM-ALFA, or long NISM-ALFA for 48 h. Nuclei were counter-stained with DAPI (blue). Scale bar, 5 μm. (B) Quantification of 5-EU staining for cells in (A) represented by violin plots. Statistical comparisons were calculated by Mann Whitney test; n = 40 cells per condition. (C) Expression analysis of 47S pre-rRNA by qRT-PCR in cells treated as in (A) represented by bar plots displaying the mean ± SD. Statistical comparisons were calculated by two-tailed Student’s t test; n = 4. (D) Immunoblot analysis of puromycin incorporation and total protein loading in WT cells transfected with EV, short NISM-ALFA, or long NISM-ALFA for 48 h. (E) Quantification of puromycin staining relative to total protein represented by bar plots displaying the mean ± SD; n = 3. (F) Representative immunofluorescence images of FBL (cyan), NISM-ALFA (green), and S9.6 (red) in cells treated as in (A). (G) Quantification of S9.6 staining for cells in (D) represented by violin plots. Statistical comparisons were calculated by Mann Whitney test; n = 25 cells per condition. (H) Representative immunofluorescence images of FBL (cyan) and NISM-ALFA/DHX9-FLAG (green) in WT cells transfected with EV, short NISM-ALFA, long NISM-ALFA or DHX9-FLAG for 48 h. (I) Quantification of nucleolar area for cells in (H) as a percentage of total nuclear area represented by violin plots. Statistical comparisons were calculated by Mann Whitney test; n = 50 cells per condition. (J) Expression analysis of 47S pre-rRNA by qRT-PCR in cells treated as in (H) represented by bar plots displaying the mean ± SD. Statistical comparisons were calculated by two-tailed Student’s t test; n = 4. Asterisks indicate: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant. All data are representative of at least two biological replicates.