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Fig. 3 | Genome Biology

Fig. 3

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

Fig. 3

MKRN1 binds at poly(A) tails. a Unmapped MKRN1 iCLIP reads display increased A-content (more than half of all nucleotides in the read), evidencing poly(A) tail binding. Cumulative fraction of iCLIP reads (y-axis, merged replicates) that could not be mapped to the human genome (see the “Materials and methods” section) and show at least a given A-content (x-axis). iCLIP data for the unrelated RBP HNRNPH [33] are shown for comparison. Cumulative percentage of reads with a minimum number of terminal A’s is displayed as an inset. b MKRN1 crosslink events increase towards 3′ UTR ends. Metaprofile of MKRN1 crosslink events and seven additional RBPs shows the normalized sum of crosslink events per nt in a 2001-nt window around annotated polyadenylation sites of transcripts with > 1 kb 3′ UTRs. Gray bars indicate windows in 3′ UTR body (− 750 to − 650) and close to the poly(A) site (− 150 to − 50), which were used to calculate enrichment factors. c MKRN1 binds near the polyadenylation site of the SRSF4 gene. Genome browser view as in Fig. 2a. d Overall RNA binding of MKRN1 is strongly reduced when abrogating PABPC1 interaction. Autoradiograph (left) of UV crosslinking experiments (replicate 1, with 4SU and UV crosslinking at 365 nm; replicates 2 and 3 in Additional file 1: Figure S6A,B) comparing GFP-MKRN1PAM2mut with GFP-MKRN1wt at different dilution steps for calibration. Quantification of radioactive signal of protein-RNA complexes and corresponding Western blot shown on the right. Uncropped gel images are shown in Additional file 3: Figure S11A,B. e MKRN1 is recruited to poly(A) RNA with the help of PABPC1. SDS-PAGE (Coomassie staining) shows recovery of recombinant His-MKRN1wt and/or His-PABPC1 (marked by petrol and gray arrowheads, respectively) from pulldown of biotinylated RNA oligonucleotides, containing the last 22 nt of the SRSF4 3′ UTR followed by 20 A (A20 RNA) or 20 V nucleotides (A or C or G; control RNA). Beads without RNA served as controls. Replicates and uncropped gel images are shown in Additional file 1: Figure S6C,D and Additional file 3: S11C-E, respectively

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