Fig. 6 | Nature Communications

Fig. 6

From: Pentatricopeptide repeat poly(A) binding protein KPAF4 stabilizes mitochondrial mRNAs in Trypanosoma brucei

Fig. 6

Distribution of KPAF4 in vivo-binding sites between pre-edited and edited mRNAs. a Isolation of in vivo KPAF4-RNA crosslinks. Modified TAP-tagged fusion protein was purified by tandem affinity pulldown from UV-irradiated (+) or mock-treated (−) parasites. The second purification step was performed under fully denaturing conditions and resultant fractions were subjected to partial on-beads RNase I digestion and radiolabeling. Upon separation on SDS–PAGE, RNA–protein crosslinks were transferred onto nitrocellulose membrane. Protein patterns were visualized by Sypro Ruby staining (left panel), and RNA–protein crosslinks were detected by exposure to phosphor storage screen (right panel). RNA from areas indicated by brackets was sequenced. Representative of six biological replicates is shown. b KPAF4 in vivo-binding sites. Crosslinked fragments were mapped to the maxicircle’s gene-containing region. Annotated mitochondrial transcripts encoded on major and minor strands are indicated by blue and red arrows, respectively. c Position-specific nucleotide frequency in partially mapped KPAF4 CLAP-Seq reads. In reads selected by partial mapping to maxicircle and edited mRNAs, the unmapped 3′ segments were considered as tail sequences. The nucleotide frequency was calculated for each position beginning from the 3′ end. d Aggregate KPAF4 mRNA-binding pattern. Read coverage is represented by the gray area, and the nucleotides in 3′ extensions are color-coded at their projected positions. e KPAF4 binding to representative pan-edited (RPS12, A6) and moderately edited (CYB) mRNAs. Read coverage profiles were created for matching pre-edited and fully edited mRNA. Read coverage is represented by the gray area, and the unmapped nucleotides in 3′ extensions are color-coded at their projected positions. The mRNA is highlighted with a rose bar in the context of adjacent maxicircle sequences. f MERS1 binding to representative pan-edited (RPS12, A6), and moderately edited (CYB) mRNAs. Graphs were created as in panel e

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