Fig 2: Proteomic analysis identifies altered abundance of select ribosomal proteins in K369I-hTau expressing primary neurons a FUNCAT-WB analysis confirms that global protein synthesis is decreased in K3 primary neurons compared to WT littermates. Primary cortical neurons were cultured from individual K3 and WT pups, before being treated with 4 mM AHA for 16 h at DIV17. The amount of new protein synthesis during this 16 h window was then quantified by using FUNCAT-WB to fluorescently tag AHA-labelled proteins. The human-tau specific Tau12 antibody was used to identify K3 positive pups. FUNCAT signal was normalised to the total protein stain REVERT. n = 4 animals, unpaired t-test. b 80 ribosomal proteins (RPs) were quantified in K3 and WT primary neurons using untargeted, label-free 1D-LC MS/MS analysis. Of these, 11 RPs (RPS2, RPS5, RPS14, RPS28, RPL5, RPL18, RPL23a, RPL35, RPL36 and MRPL12) were found to be significantly decreased (FC ≤ 0.66, p≤ 0.05) in K3 primary neurons compared to WT littermates, whereas 1 RP (RPS6) was significantly increased (FC ≥ 1.5, p ≤ 0.05) in K3 primary neurons. RPL22 is shown as an example of an RP unchanged in abundance between K3 and WT primary neurons. n = 4 animals, unpaired t-test. c Surface representation of the ribosome complex (PDB: 4ug0) with RPs found to have significantly decreased abundance in K3 primary neurons (FC ≤ 0.66, p ≤ 0.05) shown in blue, and RPS6, which displayed significantly increased abundance (FC ≥ 1.5, p ≤ 0.05) in K3 primary neurons shown in red. Unchanged RPs belonging to the 60S subunit are shown in orange, whereas unchanged RPs which form part of the 40S subunit are shown in green. d Western blot analysis reveals that the abundance of candidate RPs is decreased in 5 month-old K3 mice compared to WT littermates. Protein abundance was normalised to the total protein stain REVERT. n = 4 animals, unpaired t-test

Fig 3: P32 can be co-immunoprecipitated with mitochondrial RNase P protein.(A) RT-PCR assay for the mRNA levels of P32 or MRPP1 in siRNA treated HeLa cells. The error bars represent standard deviation of three replicates. (B) Northern hybridization for mitochondrial pre-rRNA. Total RNA prepared from HeLa cells pre-treated for 24 hrs with P32 [(−)P32] or MRPP1 [(−)MRPP1] siRNAs was analyzed by northern hybridization, using probes specific to pre-rRNA, as in Figure 5.The same blot was re-probed for 7 SL RNA, which served as a loading control. (C) Immunoprecipitation using MRPP1 antibody (MRPP1-AB) or RPL5 antibody (RPL5-AB) was performed as described in materials and methods. Co-isolated proteins were separated by SDS-PAGE, and P32 protein was determined by western analysis. (−)AB, control immunoprecipitation in the absence of antibody. (D) DNase I treatment disrupted the P32-MRPP1 interaction. Immunoprecipitation was performed using MRPP1 antibody, as in panel C. After wash, the beads were incubated with either RNase A or DNase I, to elute bound materials. The eluted proteins were analyzed by western blots for the presence of P32 protein. Buffer, 1×TE buffer alone.

Fig 4: Ablation of BRIX1 activates the nucleolar stress‐p53 pathway to suppress the growth of cancer. A‐D) Knockdown of RPL5 or RPL11 impairs the activation of p53 upon BRIX1 depletion. CAL‐51 (A, B) and HCT116 p53+/+ C, D) cells were transfected with siRNAs as indicated for 48 h, followed by IB analysis. E, F) Knockdown of BRIX1 increases the interactions of MDM2 with RPL5 and RPL11, respectively. Cells stably expressing shNC or shBRIX1 were used for co‐IP‐IB analysis with antibodies as indicated. The proteasome inhibitor MG132 was supplemented into the medium for 5 h before cell harvest. G) Knockdown of BRIX1 diminishes p53 ubiquitination induced by MDM2. HCT116 p53−/− cells stably expressing control or BRIX1 shRNA were transfected with plasmids as indicated for 48 h and treated with MG132 for 5 h, followed by in vivo ubiquitination assay and IB analysis. H) Knockdown of BRIX1 extends the half‐life of p53 protein. Cells were transfected with control or BRIX1 siRNA. Cycloheximide (CHX) (100 mg mL−1) was supplemented into the medium for the indicated time before cells were harvested for IB analysis. I, J) Knockdown of BRIX1 inhibits the proliferation of breast cancer cells with wild‐type p53. CAL‐51 (I) and MCF‐7 (J) cells were transfected with control or BRIX1 siRNAs for 6–12 h and seeded in 96‐well plates for a cell viability assay. K, L) Knockdown of BRIX1 impedes the colony‐forming ability of wild‐type p53‐harboring cancer cells. CAL‐51 (K) and MCF‐7 (L) cells were transfected with control or BRIX1 siRNAs for 6–12 h and were seeded in 6‐well plates for about 14 days. Colonies were fixed with methanol, and visualized by crystal violet staining. M, N) Knockdown of BRIX1 induces G1‐cell cycle arrest in breast cancer cells harboring wild‐type p53. CAL‐51 (M) and MCF‐7 (N) cells were transfected with control or BRIX1 siRNAs for 48 h, followed by flow cytometric analysis. O, P) Knockdown of BRIX1 represses the migration of wild‐type p53‐harboring cancer cells. CAL‐51 (O) and MCF‐7 (P) cells were transfected with control or BRIX1 siRNAs for 6–12 h, followed by a cell migration assay. ***p < 0.001.

Fig 5: Ablation of UTP11 stabilizes p53 through RPL5/RPL11 inhibition of MDM2. (A–D) Knockdown of RPL5 or RPL11 compromises the induction of p53 by UTP11 depletion. CAL-51 (A, B) and HCT116 p53+/+ cells (C, D) were transfected with control, UTP11 siRNA, RPL5 siRNA, and RPL11 siRNA as indicated. Cell lysates were subjected to IB analysis with indicated antibodies. (E, F) RPL5-MDM2 and RPL11-MDM2 interactions are increased by depletion of UTP11. CAL-51 cells were transfected with control or UTP11 siRNA, followed by co-IP-IB assays using antibodies as indicated. The proteasome inhibitor MG132 was supplemented into medium for 5 h before cell harvest. (G) Knockdown of UTP11 diminishes MDM2-induced p53 ubiquitination. HCT116 p53−/− cells stably expressing control or UTP11 shRNA were transfected with plasmids encoding p53, His-Ub, and HA-MDM2 as indicated and treated with MG132 for 5 h, followed by in vivo ubiquitination assay and IB analysis. (H) UPT11 knockdown extends the half-life of p53 protein. CAL-51 cells were transfected with control or UTP11 siRNA. CHX (100 mg/ml) was supplemented into medium for the indicated time before cells were harvested for IB analysis. ***p < 0.001.