Fig. 5
MKRN1 ubiquitylates ribosomal protein RPS10 and translational regulators. a Ubiquitin remnant profiling to compare the relative abundance of ubiquitylation sites in MKRN1 KD2 and control HEK293T cells. Ubiquitin remnant peptides were enriched and analyzed by quantitative mass spectrometry, quantifying a total of 15,528 ubiquitylation sites on 4790 proteins. 29 putative MKRN1 target sites with significantly decreased ubiquitylation upon MKRN1 KD2 (FDR < 10%, n = 4 replicates) are highlighted and labeled with the respective protein name. Note that many proteins contain several differentially regulated ubiquitylation sites. b Protein interaction network of 21 proteins with putative MKRN1 ubiquitylation target sites (significantly reduced, shown in a). The functional interactions were obtained from the STRING and BioGrid databases and our study. Visualization by Cytoscape. c Ubiquitin remnant profiling results for significantly regulated ubiquitylation sites (FDR < 10%) in proteins from network in b. Mean and standard deviation of the mean (s.d.m., error bars) are given together with all data points. d Comparison of interactome of GFP-MKRN1wt (WT over GFP, see Fig. 1a) with putative MKRN1 ubiquitylation substrates from ubiquitin remnant profiling (UB, see a). Protein names are given for all ubiquitylation substrates. e Ubiquitin remnant profiling results for seven quantified ubiquitylation sites in RPS10 and RPS20. Significant changes are shown in black (FDR < 10%) and non-significant changes in gray. Representation as in c. f Comparison of ubiquitylation sites in the target proteins RPS10 (UniProt ID P46783), RPS20 (P60866), PABPC1 (P11940), PABPC4 (B1ANRO), IGF2BP1 (Q9NZI8), IGF2BP2 (F8W930), and IGF2BP3 (O00425) that are modified by ZNF598 and MKRN1 during RQC. g MKRN1 ubiquitylates RPS10 and PABPC1 in vitro. His-RPS10 (left) or His-PABPC1 (right) were incubated with or without His-MKRN1. Ubiquitylation of the target proteins was assessed by Western blot. Replicates 2, 3, and uncropped gel images are shown in Additional file 3: Figure S12I,J for RPS10 and Additional file 3: Figure S12K,L for PABPC1