Skip to main content

Advertisement

Fig. 1 | Genome Biology

Fig. 1

From: The RNA-binding ubiquitin ligase MKRN1 functions in ribosome-associated quality control of poly(A) translation

Fig. 1

MKRN1 interacts with PABPC1 and other regulators of translation and RNA stability. a Protein interactome of GFP-MKRN1wt in HEK293T cells analyzed by quantitative MS-based proteomics. Combined SILAC ratios (n = 3 replicates) after z-score normalization are plotted against log10-transformed intensities. 1100 protein groups were quantified in at least two out of three replicate experiments. MKRN1 and significant interactors are highlighted (FDR < 5%). b MKRN1 and PABPC1 associate with polysomes. A 10–50% sucrose gradient of cycloheximide-treated HEK293T cell extracts. Shown are the Western blot analyses of individual gradient fractions with antibodies against MKRN1 and PABPC1/3 (n = 3 replicates, plus one technical replicate). UV absorbance was measured at λ = 254 nm. Replicates and uncropped gel images are shown in Additional file 3: Figure S10A-C. c A PAM2 motif similar to the previously reported consensus (shown on top; Additional file 1: Figure S1B) [22] is present in MKRN1 (first amino acid position indicated on the left). Introduced mutations in MKRN1PAM2mut are indicated in petrol below. Relevant positions are highlighted (Additional file 1: Figure S1B). d Endogenous PABPC1 interacts strongly with MKRN1wt and MKRN1RINGmut, but only to a lesser extent with MKRN1PAM2mut. Western blots for endogenous PABPC1 and GFP after AP of GFP-MKRN1 (wt and mutants). Ratios of PABPC1 signal (normalized to input) in GFP-MKRN1 APs over control (GFP empty vector, EV) are shown below. Replicates 2, 3, and uncropped gel images are shown in Additional file 3: Figure S10D-F. e Quantitative comparison of the interactomes of GFP-MKRN1wt and GFP-MKRN1PAM2mut shows that PABPC1 and several other interactors are lost upon PAM2 mutation. Combined ratios of three replicates are shown in a scatter plot. Only proteins detected in at least two out of three replicates are shown. MKRN1wt significant interactors (from a) are highlighted as in a (FDR < 5% in MKRN1wt). f MKRN1WT and MKRN1PAM2mut, but not MKRN1RINGmut, efficiently autoubiquitylate. In vitro ubiquitylation assays with recombinant His-tagged MKRN1wt and mutant proteins that were incubated with or without the E2 enzyme UBC5a, the E1 enzyme UBA1, and ubiquitin. A reaction with UBC5a only served as a control. Autoubiquitylation was analyzed by Western blot. Replicates 2, 3, and uncropped gel images are shown in Additional file 3: Figure S10G,H

Back to article page