Fig 2: Expression of PRMT5 and WDR77 in breast cancer (A). Expression analysis of 109 paired samples from The Cancer Genome Atlas (TCGA) database shows significant overexpression of PRMT5 and WDR77 in breast cancer samples relative to matched normal samples (B). Fold expression of WDR77 and PRMT5 in breast normal (MCF10A), ER+ (MCF7, T47D) and ER- (MDA-MB-231, HCC38) breast cancer cell lines. Normalized to GAPDH (C). Immunoblotting of WDR77 and PRMT5 in nuclear and cytoplasmic extracts of breast normal and cancer cell lines. Actin was used as loading control. (D). Fold expression (relative to GAPDH) of WDR77 and PRMT5 and (E). Immunoblotting of the respective proteins in sh Scrambled, sh WDR77 and sh PRMT5-treated samples. Actin was used as loading control in E (F). (Top panel) Annexin V staining showing increased number of apoptotic cells upon loss of WDR77 and PRMT5. (Bottom panel) Box plots of results from apoptosis assay for three biological replicates. Student's t-test *P < 0.05 **P < 0.005 ***P < 0.0005.

Fig 3: Alternative splicing coupled mRNA decay of transcripts upon loss of PRMT5. qPCR analysis showing (from left to right) relative levels of exon inclusion, pre-mRNA and the mRNA/pre-mRNA ratio in sh Scrambled and sh PRMT5 samples for (A) REPIN1/AP4 (B) ST3GAL6 (C) PFKM (D) TRNAU1AP/SECP43. The illustrations depict the gene structure with exons shown as black boxes and introns as lines. The included exons are shown in red. Arrows indicate regions to which primers were designed. Student's t-test *P < 0.05 **P < 0.005 ***P < 0.0005.

Fig 4: ZNF326 is symmetrically dimethylated at R175 by the PRMT5/WDR77 complex (A). ZNF326 has two glycine-arginine rich motifs. Asterisk indicates R175 that was identified to be dimethylated (B). Immunoblots of immunoprecipitates showing symmetric dimethylation of ZNF326 (C). Representative tandem mass spectrum of the peptide GR(28.0314)GTPAYPESTFGSR {m/z:537.602 (+3). The fragment ion matching within 10 ppm are shown as either the B-ion (purple) or Y-ion (blue) series. The green dashed line indicates precursor m/z. The dotted lines in the fragmentation ladder sequence on the top the spectrum corresponds to the missing B-ion (purple) and Y-ion (blue) series (D). Mass Spectrometry analysis showing the relative abundance of random and dimethylated peptides in cells infected with sh Scrambled, sh WDR77 and sh PRMT5 and (E). Graphical representation of the same.

Fig 5: Networks of top interaction partners of WDR77 in the cytoplasm and nucleus identified by LC-MS/MS (A). Gene ontology analysis (B). Protein interaction network of top 15 interacting partners of WDR77 in the cytoplasm (red lines: newly identified interactions, black lines: previously characterized interactions) (C). Gene ontology analysis (D). Protein interaction network of top 15 interacting partners of WDR77 in the nucleus (red lines: newly identified interactions, black lines: previously characterized interactions) (E). Immunoblots of co-immunoprecipitations of WDR77, PRMT5 and ZNF326 (IP-Immunoprecipitation, WB-western blot).