Fig 2: GREB1 stabilizes ERa by limiting binding of the ubiquitin ligase ZNF598.(A to E) MCF7-WT or GREB1-KO cells were transduced with FLAG-ERa, and FLAG pulldown was performed. Eluates were analyzed by MS. The number of peptide reads for ERa (A) and GREB1 (B) was examined as controls. The number of peptide reads for ubiquitin (C), unique or common interactors (D), and ZNF598 (E) from MCF7-WT and GREB1-KO was illustrated. (F) FLAG pulldown was performed to MCF7-WT or GREB1-KO cells transduced with ERa-WT or ERa-2M. (G) Densitometry analysis of ZNF598 bands from the Western blot in (F) for corresponding lanes. n = 3; ***P < 0.005 by one-way ANOVA.

Fig 3: MKRN1 stalls ribosomes at poly(A) sequences. a The dual fluorescence reporter harbors an N-terminal GFP, followed by a FLAG-SR-X linker and a C-terminal RFP, which are separated by P2A sites to ensure translation into three separate proteins [8]. The resulting GFP:RFP ratio was determined using flow cytometry. The inserted fragment K(AAA)20 encodes 20 lysines by repeating the codon AAA. The starting vector without insert (K0) served as control. Schematic ribosomes illustrate translation of the respective reporter segments. b Ribosomes fail to stall in the absence of MKRN1. HEK293T cells were transfected with control siRNA or siRNAs targeting MKRN1 (KD1 and KD2) or ZNF598 for 24 h, followed by transfection of the reporter plasmids for 48 h. Western blots for KDs are shown in Additional file 1: Figure S8A. RFP and GFP signals were analyzed by flow cytometry. Median RFP:GFP ratios, normalized to K0 in control, are shown. Error bars represent s.d.m.; P values indicated above (paired two-tailed Student’s t test, Benjamini-Hochberg correction, n = 6 replicates; ns, not significant). Analyses for inserts coding for 12 lysines (K(AAA)12) and ten arginines (R(CGA)10) in the dual fluorescence reporter are shown in Additional file 1: Figure S7A. c Expression of MKRN1wt can rescue ribosome stalling. HEK293T cell lines with stable integrations of siRNA2-insensitive MKRN1 wild type and mutant constructs, or empty vector, were transfected with MKRN1 siRNA2 for 24 h, followed by transfection of the reporter plasmids for 48 h. RFP and GFP signals were analyzed by flow cytometry. Median RFP:GFP ratios, normalized to K0 in WT cells, are shown. Error bars represent s.d.m.; P values indicated above (paired two-tailed Student’s t test, Benjamini-Hochberg correction, n = 6 replicates; ns, not significant). Analyses for reporter plasmids with inserts coding for K(AAA)12 or R(CGA)10 are shown in Additional file 1: Figure S7B. d MKRN1 knockout (MKRN1 KO) and wild type (WT) HEK293T cells were transfected with the reporter plasmids for 48 h. Measurements, analyses, and visualization as in c (n = 4 replicates). Analyses for reporter plasmids with inserts coding for K(AAA)12 or R(CGA)10 are shown in Additional file 1: Figure S7C (d)

Fig 4: ZNF598 Ubiquitinates eS10 In Vitro and In Vivo(A) In vitro ubiquitination reactions of ribosomes using HA-tagged ubiquitin and the indicated factors were analyzed by immunoblotting with anti-HA to detect all new ubiquitin conjugates. The last reaction was also separated on a 10%–50% sucrose gradient and analyzed by immunoblotting for the indicated antigens (right panel). Asterisks indicate primary ribosomal ubiquitin conjugates.(B) Ribosome ubiquitination reactions with His-tagged ubiquitin in the presence (+) or absence (-) of ZNF598 were fractionated to purify ubiquitinated core ribosomal proteins, treated or untreated with the catalytic domain (CD) of the deubiquitinase Usp2, and analyzed by SDS-PAGE and Coomassie blue staining. The major ZNF598-dependent products were identified by mass spectrometry.(C) Ribosomes were ubiquitinated with increasing concentrations of ZNF598 with either HA-tagged ubiquitin or methyl-ubiquitin, and analyzed by immunoblotting for the indicated ribosomal proteins.(D) eS10 ubiquitination status in WT and ZNF598 KO cells was analyzed by immunoblotting. Two exposures are shown.(E) WT or ZNF598 KO cells were transfected with HA-ubiquitin and treated with nothing, 100 µg/ml cycloheximide, or 1 mM DTT for 2 hr. Anti-eS10 immunoblots are shown for total cell lysate and anti-HA affinity purified products. The positions of ubiquitin- or HA-ubiquitin-modified eS10 are indicated. See also Figure S3.

Fig 5: ZNF598 Is Required to Initiate the RQC Pathway during Poly(A) Translation(A) (KAAA)21 cells were transfected with control (blue) or ZNF598 (red) siRNAs for 72 hr, reporter expression induced for 24 hr, and analyzed by flow cytometry (left and middle panels) or immunoblotting for the indicated proteins (right panel). (K)0 cells (gray) served as a reference.(B) (KAAA)21 cells were subjected to the indicated siRNA treatment for 72 hr, reporter expression induced for 24 hr, and analyzed by immunoblotting for the indicated proteins.(C) (KAAA)21 reporter expression was induced for 24 hr in wild-type (WT, in blue) and ZNF598 knockout (KO, in red) cells and analyzed by flow cytometry.(D) (KAAA)21 cells knocked out for ZNF598 were transiently transfected with the indicated plasmids and the transfected cells (as judged by a co-transfected BFP construct) were analyzed by flow cytometry after reporter induction for 24 hr. The majority of cells displayed restoration of terminal ribosomal stalling to wild-type levels (circled). See also Figure S2.