Fig 2: C9orf72 associates with the E3 ligase UBR5 and BAG6 chaperone complex AExperimental workflow to identify C9orf72 quality control factors.BVolcano plot of C9orf72 candidate interacting proteins upon proteasomal inhibition. Lysates from C9orf72‐HA expressing SMCR8 KO cells treated with DMSO or Btz were subjected to HA‐IP and C9orf72 immune complexes were analyzed by mass spectrometry. n = 4 biological replicates with each including n = 2 technical replicates. Pearson's correlation, DMSO ≥ 0.747, Btz ≥ 0.873. Interacting candidates reaching FDR < 0.01, s0 = 1 (Student's t‐test) are highlighted in dark gray.CC9orf72‐HA expressing SMCR8 KO cells were treated with DMSO or Btz and analyzed by immunoblotting. α‐tubulin served as loading control.DLysates from C9orf72‐HA expressing parental and SMCR8 KO cells treated with DMSO or Btz were subjected to HA‐IP followed by immunoblot analysis. Empty parental and SMCR8 KO served as controls.ESMCR8 KO cells expressing C9orf72‐HA were transiently transfected with GFP‐UBR5 or GFP and treated with DMSO or Btz. Lysates were incubated with Streptavidin agarose coupled with biotinylated GFP VHH nanobodies and bound proteins were analyzed by immunoblotting.FParental 293T cells were transiently transfected with GFP‐UBR5 or MOCK followed by pulldown and immunoblot analysis. Data information: NRF2 confirmed proteasomal inhibition by Btz. MOCK refers to control transfection without plasmid. Source data are available online for this figure.

Fig 3: Interaction of BAG6 and UBR5 with uncomplexed C9orf72 and SMCR8 A293T SMCR8 KO and C9orf72 KO cells were transfected with GFP‐C9orf72 and GFP‐SMCR8, respectively. GFP served as control. Cells were lysed under mild conditions, lysates were incubated with biotinylated GFP VHH coupled with Streptavidin agarose and analyzed by Western blot.BSMCR8 KO cells stably expressing GFP or GFP‐C9orf72 were treated with Btz and subjected to streptavidin pulldown using biotinylated GFP VHH nanobodies under denaturing conditions. Source data are available online for this figure.

Fig 4: UBR5 is a modulator of MYC stability. (A) Gene enrichment score in high GFP/mCHERRY-sorted cells relative to unsorted cell population on day 7 of library infection visualized as LogFC. (B) sgRNA-mediated knockout of UBR5 (sgUBR5), FBXW7 (sgFBXW7) or a non-targeting sgRNA control (sgNT) in B6 clone and analysis of GFP, mCHERRY, and GFP/mCHERRY ratio using FACS 7 days post-knockout. (C) MYC degradation screen using an arrayed sgRNA sublibrary. MYC-GFP/mCHERRY cells were infected with lentiviruses encoding for sgRNAs targeting HECT E3 ligases. 2 sgRNAs were used per gene (indicated as 1, 2). Seven days after infection and selection with puyromycin, GFP/mCHERRY was measured using flow cytometry and the increase in GFP/mCHERRY ratio upon knockout of the singlegenes was compared to a non-targeting control (sgNT).

Fig 5: Validation of UBR5 as a regulator of MYC protein stability and interaction. (A) B6 clone was transfected with sgNT or sgUBR5 for 7 days, followed by treatment with cycloheximide at 20 µM concentrations. MYC-GFP level were assessed by western blot at the indicated time points (quantification is shown in Supplementary Fig. 4A). (B) Increase in the protein level of MYC-wt as well as MYC-GFP fusion protein upon UBR5 knockdown in HEK293A cells (WT) and B6 clone (MYC-GFP-mCHERRY). (C) qPCR analysis of MYC target genes upon knockdown of UBR5. (D) Immunoprecipitation analysis showing interaction between UBR5 and MYC in B6 clone. (E) Decrease in ubiquitination of MYC-GFP immunoprecipitated protein upon knockout of UBR5 and FBXW7. Relative decrease in K48 ubiquitination was normalized to sgNT (1) and values are indicated on the bottom of the corresponding lane. (F) B6 clone was transfected with sgNT or sgUBR5 for 4 days and then further transfected with vector expressing mock DNA or UBR5 ORF, respectively to rescue the phenotype. GFP/mCHERRY was measured 72 h after transfection with rescue construct. (G) Ubiquitination assay utilizing UBR5(2463–2792) and peptide substrates MYC_174–196_KK (Fluo-SSLYLQDLSAAASECIDPSVVFPKK) and MYC_174–196 (SSLYLQDLSAAASECIDPSVVFP) resolved on a 5–20% SDS-PAGE gel. Fluorescein fluorophore was detected on a ChemiDoc System (Bio-Rad). (H) Ubiquitination assay utilizing UBR5(2463–2792) and the conservatively mutated peptide substrate MYC_174–196_KKMut (Fluo-SSLYLQDVTAAATDSLDPSVVFPKK), in which mutated residues are highlighted in bold. Samples were resolved on a 5–20% SDS-PAGE gel and fluorescence was detected on a ChemiDoc System (Bio-Rad).