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. 2021 Nov 23;22(23):12639. doi: 10.3390/ijms222312639

Figure 6.

Figure 6

CAF1 and CAF2 promote the binding of SOT1 to the 5′ end of ndhA transcripts. (a) Electrophoretic mobility shift assay (EMSA) showing the positive effect of CAF1 and CAF2 on the binding of SOT1 to the 5′ end of ndhA transcripts. The predicted binding sequence (UGGCUGAUAUUA) of SOT1 on ndhA transcripts was synthesized and labeled with biotin. Increasing concentrations of recombinant CAF1 or CAF2 proteins were incubated with 10 nM biotin-labeled probe and the MBP-SOT1 protein. The competitor used in the competition experiments was an unlabeled probe corresponding to the predicted binding sequence of SOT1 on the ndhA transcripts. The asterisk indicates an unspecific band. Three independent experiments were performed, and one representative experiment is shown. (b) Overexpressing CAF proteins promoting the binding of SOT1 to the 5′ end of ndhA transcripts in vivo. The HA-tagged CAF1 and FLAG-tagged CAF2 were overexpressed (OE) in protoplasts isolated from 12-day-old seedlings. Chloroplasts were harvested from the transfected protoplasts. Protein complexes were immunoprecipitated (IP) using an α-SOT1 antibody. (Left) Immunoblot analysis of the protein presence in chloroplast extract as immunoprecipitated using an α-SOT1 antibody. The immunoblot results showed a comparable enrichment of SOT1 protein in samples. (Right) The relative levels of psbA, psbB, and ndhA 5′-end mRNAs in the α-SOT1 immunoprecipitated complexes were determined using a qPCR. Student’s t-test was carried out to determine the significance of the fold enrichments of SOT1 on ndhA 5’-end RNAs between WT and OE.CAF protoplast. ** indicates a significant difference at p < 0.01. Mean values ± SD of the triplicate replicates are shown. (c) The loss of CAF proteins leads to the decreased association of SOT1 with the 5′ end of ndhA transcripts. The stromal extracts from wild-type, caf1, caf2, and caf1 CAF2-interference (RNAi) plants were immunoprecipitated with an α-SOT1 antibody. (Upper) Immunoblot analysis of the protein presence in the chloroplast extract, as immunoprecipitated using an α-SOT1 antibody. (Lower) The relative levels of psbA, psbB, and ndhA 5′-end mRNAs in the α-SOT1 immunoprecipitated complexes were determined using qPCR. Mean values ± SD of the triplicate replicates are shown.