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Fig. 4 | Genome Biology

Fig. 4

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

Fig. 4

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)

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