Fig 2: (A) Six residues in peptide 29 (X1–X6) were modified one at a time and replaced with one of the listed amino acids. (B) List of modified sequences with improved affinity for PRMT5-MEP50 over 29.

Fig 3: PRMT5 Promotes Homologous Recombination-Mediated DSB Repair Independently of ATM Activation(A) HeLa-shCTRL and shPRMT5 cells were stimulated with 3Gy IR and RAD51 foci analyzed 6 hr later. Quantification of number of cells with >10 RAD51 foci is shown below (mean ± SEM; **p = 0.006; n = 3).(B) Quantification of RPA foci in CENPF-positive (late S/G2) cells 8 hr after 3Gy IR is shown (mean ± SEM; **p = 0.006; n = 3).(C) Time course of phosphorylated and total protein levels is shown.(D) U2OS-DR3 cells were treated with siRNA as indicated before transfection of I-Sce1 and myc-PRMT5. The number of GFP-positive cells (HR proficient) was determined by flow cytometry (mean ± SD; n = 3).(E) PRMT5-depleted cells were treated with increasing doses of camptothecin (CPT; left panel) or Olaparib (right panel), and survival was determined by colony assay (mean ± SD; n = 3).(F) Cells were treated with CPT (1 µM) for the time points indicated and immunoblotted for phosphorylated and total proteins. Scale bar, 10 µm. See also Figure S2.

Fig 4: Effects of type I PRMT inhibition in human PBMCs. Western Blot analysis of monomethyl-arginine (MMA), symmetric dimethyl arginine (SDMA), asymmetric dimethyl arginine (ADMA), PRMT1 and PRMT5 (NC, non-cultured, NS, non-stimulated, TCRact, T Cell Receptor activated) (A) and RT-PCR for several PRMTs (B) in non-stimulated and TCR-activated human PBMCs from healthy donors treated with either DMSO or 2 µM GSK3368712 for 72 h (two-tailed Student’s t test: *p < 0.05, **p < 0.01, ***p < 0.001; n.s., not significant). (C–E) MethylScan analysis of arginine methylation in human PBMCs treated with GSK3368712. (C) Venn diagram representation of proteins modulated by the type I PRMT inhibitor at MMA, ADMA and SDMA sites across four healthy donors. (D) Common set of proteins where a decrease in ADMA and concomitant increase in MMA and SDMA was observed in all 4 donors. (E) Pathway analysis of all proteins that underwent any change (> 2.5 fold) in arginine methylation upon treatment with GSK3368712, compared to DMSO. FDR, False Discovery Rate.

Fig 5: (A) FP results for linear sequence 29 and 2nd and 3rd generation macrocyclic PAPIIs with PRMT5-MEP50 (n = 3). (B) FP competition of representative linear and macrocyclic peptides with FITC-labeled 50 for binding to PRMT5-MEP50 (n = 3). (C) FP competition of 2nd and 3rd generation PAPIIs and the unlabeled pICln protein with the Alexa 488-labeled full-length human pICln protein for binding to PRMT5-MEP50 (n = 3). (D) Comparison of the effects of linear and cyclic peptides on the melting temperature of the TIM-MEP50 complex, as determined by TSA (n = 6). (E) Pull-down assay with peptides 55 and scrambled 56 immobilized on the DBCO beads using MCF7 cell lysate. Immobilized 55 was also tested with the lysate containing 100 µM of free 56. (F) GFP-IP from the lysate of Flp-In T-REx 293-GFP and Flp-In T-REx 293-GFP-PRMT5 overexpressing cells, testing compound 53 and scrambled 54 at 50 µM. (G) GFP-IP from Flp-In T-REx 293-GFP-pICln and Flp-In T-Rex 293-GFP-RioK1 cytoplasmic extract, testing 53 and 54 at 50 µM.