Fig 2: RPL11 and RPL5 inhibits breast cancer cell proliferation and induced apoptosis.a Bioinformatics analysis of RPL11 and RPL5 expressions in breast cancer and para-carcinoma tissues based on TCGA data. b Kaplan–Meier curves of breast cancer survival by RPL11 and RPL5 expressions based on TCGA data. c Expressions of Bcl2, P53, Bax, CDK2, P21, RPL11, and RPL5 in cells transfected with RPL11 or RPL5 overexpression vector. d Immunofluorescence of FLAG-tagged plasmids expressing RPL11 and RPL5 in MCF7 and ZR-75-1 cells. The nuclei were stained by DAPI. e MTT assay of cell proliferation at 24, 48 and 72, 96 h after transfection with RPL11 or RPL5 overexpression vector. *P < 0.05, **P < 0.01. f Colony formation assay 14 days after transfection. *P < 0.05. g Flow cytometry analysis of cell cycle in MCF7 and ZR-75-1 cells. *P < 0.05, **P < 0.01. h Flow cytometry analysis of cell apoptosis after transfection with RPL11 or RPL5 overexpression vector. *P < 0.05, **P < 0.01.

Fig 3: Hydrophobic surfaces impact Rad6’s association to ribosomes(A) Molecular docking predicts the binding poses of Rad6 on the ribosome for two ubiquitination sites on eS12 (K85 and K90). In the blowouts, the ribosome receptor sites are shown as a surface and are colored tan/cyan for hydrophobic/hydrophilic residues. Details are provided for the predicted contact sites. Additional views for these interaction surfaces are present in Figure S4F.(B) Schematic of Rad6 constructs with respective name notations. Green represents the C-terminal last alpha helix, blue represents the C-terminal acidic tail, and red represents the acid region DEE60-62.(C) Immunoblot from sucrose density sedimentation analysis shows differential ribosomal association pattern for Rad6LLL variant. Anti-uS3 and -uL5 were used as markers for the 40S and 60S ribosome subunits, respectively.(D and E) Anti-HA (Rad6) immunoblot after sucrose cushion for Rad6 variants. Rad6 LLL and Rad6 N94L (D) and Rad6 N84A, Rad6 N84L, Rad6Y82A, and Rad6Y82E (E) have an increased presence in the ribosome pellet. Anti-HA (Rad6) was used to determine expression levels of Rad6 variants in the whole-cell lysate, anti-GADPH was used as loading control for whole-cell lysate, and anti-uS3 was used as ribosomal loading control.(F) Immunoblot from sucrose density sedimentation analysis shows reduced ribosomal co-sedimentation for Rad61–149 variant. Same gel comparison is presented in Figure S5A.(G) Immunoblot from supernatant (open circle) and ribosome pellet (full circle) after sucrose cushion from strains expressing Rad61–149 and Rad6LLL variants. uS10 was used as ribosome marker.(H) Immunoblot from sucrose density sedimentation analysis shows reduced ribosomal co-sedimentation for Rad61–122 variant. Same gel comparison is presented in Figure S5B.

Fig 4: Rad6 co-sediments with elongating ribosomes(A) Immunoblot of co-immunoprecipitation (coIP) of Rad6-FLAG with ribosomal proteins uS3 and uL5.(B) Rad6 presence on the ribosome pellet is independent of cellular lysate incubation with 10 mM DTT, 1 M NaCl, 1 mM H2O2, and DNAse (0.1 U/µg protein) and RNAse I (100 U/µg protein). 1% SDS was used to disrupt molecular interactions. Anti-uS3 and -uL5 were used as loading control for proteins from the 40S and 60S ribosome subunits, respectively.(C) Sucrose density sedimentation analysis and anti-HA immunoblot of lysates from cells expressing Rad6-HA, GFP-HA, or GFP-Rad6-HA constructs. Anti-uS3 and -uL5 were used as markers for the 40S and 60S ribosome subunits, respectively.(D) Purified Rad6, but not GFP, relocalizes to the polysomal fraction after incubation with ribosomes for 10 min at room temperature followed by sucrose density-sedimentation separation. Ribosomes were isolated from rad6?bre1? strain. Anti-uL5 was used as a marker for the 60S ribosome subunit. *Anti-Rad6 antibody unspecific band.(E) 3D classification of ribosomes incubated with purified Rad6 (RiboRad6mix) and ribosomes co-immunoprecipitated with FLAG-tagged Rad6 (RiboRad6IP). Ribosomes were observed in the pre-translocation (PRE), post-translocation (POST), and transition (PRE-to-POST) states.(F) Structural detail of RiboRad6IP at the pre-translocation stage with tRNAs in hybrid A/P and P/E positions. Rigid fit of the tRNAs was performed using coordinates from the 80S ribosome (PDB: 6GZ5) (Flis et al., 2018).(G) Focused classification of RiboRad6IP shows two classes with distinct conformations for the 40S beak and ribosomal proteins eS12 and eS31. The extended beak rRNA model was built using COOT (Emsley and Cowtan, 2004).

Fig 5: MeCP2 promotes breast cancer cell proliferation through regulating P53 degradation by inhibiting the expression of RPL11 or RPL5.a MTT assay. *P < 0.05, **P < 0.01. b Cell colonies assay. *P < 0.05, **P < 0.01. c Cell cycle analysis. *P < 0.05, **P < 0.01. d Ubiquitination assay. e The P53 protein levels in MCF7 cells co-transfected with MeCP2 and RPL11 or MeCP2 and RPL5, and in cells transfected with MeCP2 overexpression vector and treated with MDM2 inhibitor (Nutlin3). f MTT assay. *P < 0.05, **P < 0.01. g Cell colonies. *P < 0.05, **P < 0.01. h Cell apoptosis assay. *P < 0.05, **P < 0.01. i Cell cycle analysis in MCF7 cells co-transfected with MeCP2 siRNA and RPL11/ RPL5 siRNA. *P < 0.05, **P < 0.01. j P53 protein expressions in MCF7 cells transfected with MeCP2 siRNA, RPL5 siRNA, or RPL11 siRNA, or treated with Nutlin3.