The N- and C-terminal domains of designer pentatricopeptide repeat proteins are dispensable for <i>in vivo</i> stabilization of chloroplast RNAs

Artificial pentatricopeptide repeat (PPR) proteins (called as well designer PPR or dPPRs) are customized RNA binding proteins made of tandem repeats of a consensus 35 amino acid motif whose RNA base recognition can be programmed by the use of a two amino acid code. Recently, we designed an artificial PPR protein called dPPRrbcL based on a PPR consensus repeat scaffold flanked by N-terminal and C-terminal domains (NTD and CTD) derived from the native maize protein PPR10, and successfully expressed this protein in Arabidopsis chloroplasts to stabilize a processed 5'-end of rbcL mRNA. While the PPR repeats in dPPRs are expected to confer RNA binding and protection from exoribonuclease, the importance of the N-terminal and C-terminal domains for dPPR in vivo activity remains unknown. Here, we used functional complementation assays in Arabidopsis using truncated versions of dPPRrbcL to examine the contribution of the NTD and CTD to rbcL mRNA stabilization in chloroplasts. The results showed that the NTD and CTD are not required for the in vivo stabilization of the processed 5' end of rbcL mRNA by dPPRrbcL but the NTD likely protects a few nucleotides at the 5'-end of the RNA sequence bound by the PPR motifs against the action of exoribonucleases. These discoveries indicate that a PPR repeat scaffold itself is sufficient to efficiently stabilize processed RNAs in chloroplasts.