Fig 2: Knockdowns of U2AF2 CIMs and PIMs perturbs activation marker expression, cell proliferation and cytokine secretion. (A) RT-qPCR validation of siRNA knockdown for U2AF1, SYNCRIP, ILF2, and SRRM2. Error bars represent mean ± S.D. (n = 6). Immunoblot analysis shows reduced protein levels for U2AF1 with protein expression quantified and normalized to tubulin (representative experiment). (B) Histogram depicts expression of the cell activation markers CD25 and CD62L from FACS analysis of resting and activated T cells treated with negative control siRNA (blue) or U2AF1 siRNA (green). (C) Proliferation of resting and activated CD4 T cells after siRNA treatment as measured by MTT assays. Error bars represent mean ± S.D. (n = 4). (D) ELISA results for secretion of 11 cytokines after knockdown of U2AF2 interactome members plotted as log2 fold changes. Error bars represent mean ± S.E.M. (n = 3). Statistical significance determined by Student’s t-test (*: p <0.05).

Fig 3: High NUSAP1 and ILF2 expression correlated with DNA repair pathways and adverse clinical features in prostate adenocarcinoma. (A) NUSAP1 mRNA levels were significantly higher in prostate adenocarcinoma compared to non-cancerous prostate tissues in the TCGA PRAD dataset. (B) GSEA demonstrated NUSAP1 mRNA level was positively correlated with the DNA repair pathway. (C) Heatmap of genes showing high correlation with NUSAP1 transcript levels. (D) NUSAP1 mRNA levels are associated with tumor grade (Gleason scores) while ILF2 and DHX9 mRNA levels were less correlated with tumor grade. (E) Kaplan–Meier analysis shows that increased NUSAP1 and ILF2 transcript levels, divided at the median, are associated with disease-specific survival; DHX9 expression levels are not associated with worse outcomes (p-values by Log-rank test). (F) Pearson correlation coefficient analysis revealed that NUSAP1 mRNA level was significantly correlated with DHX9 and ILF2 mRNA levels. ****, p < 0.0001; NS, p > 0.05.

Fig 4: NUSAP1, ILF2, and DHX9 interact with R-loops. (A) Venn diagram of 33 proteins found in both NUSAP1 and RNA/DNA hybrid interactomes [41], including ILF2 and DHX9. (B) Gene ontology (GO) analysis of the 33 common NUSAP1 and R-loop-interacting proteins shows enrichment for RNA and telomerase RNA binding. (C) Immunoprecipitation with S9.6 antibody that binds specifically to RNA/DNA hybrids confirms interactions with DHX9, Flag-NUSAP1, ILF2, and HNRPC. Binding was abolished by pretreatment with RNase H, confirming specificity of their interactions with R-loops. (D,E) Confocal microscopy images of pre-extracted LNCaP cells for native NUSAP1, DHX9, nucleolin, and R-loops (S9.6) demonstrate colocalization. Scale bar = 10 µm.

Fig 5: Identification of NUSAP1 interactors, ILF2, and DHX9 by AP–MS. (A) Flowchart of AP–MS and verification of protein-protein interactions by co-IP and immunofluorescence. (B) Venn diagram of 85 Flag-NUSAP1 interacting proteins identified by AP–MS. Nocodazole induced mitotic arrest of 58 mitosis-enriched proteins while non-synchronized cells (vehicle) identified 81 proteins of which 27 interactors were not mitosis associated. (C) Gene ontology (GO) analysis of the molecular functions (MF) of the 27 non-synchronized NUSAP1 interactors by DAVID are enriched for RNA and DNA binding. (D) Ingenuity Pathway Analysis (IPA) reveals NUSAP1 interaction with an ILF2/DHX9 protein network. (E) Significant overlap 8 interactors between the NUSAP1 and ILF2 interactomes [40] including ILF2 and DHX9. (F) Gene ontology (GO) analysis of the molecular functions (MF) of the 8 common interactors between NUSAP1 and ILF2 interactomes by DAVID are enriched for RNA and DNA binding. (G) Immunoprecipitation and western blots validate DHX9, ILF2, and HNRPC interactions with Flag-NUSAP1. (H) Confocal microscopy shows ILF2 and DHX9 under conventional and pre-extraction conditions. For pre-extraction, cells were permeabilized by 0.5% Triton X-100 before PFA fixation. Under pre-extraction, ILF2 and DHX9 are visualized mainly in the nucleolus, shown by staining with nucleolin. (I) NUSAP1 and DHX9 co-localize with nucleolin in LNCaP cells. Arrowheads indicate mitotic cells. Scale bar = 10 µm.