A Moss Pentatricopeptide Repeat Protein Binds to the 3' End of Plastid ClpP Pre-mRNA and Assists with MRNA Maturation

Pentatricopeptide repeat (PPR) proteins constitute a large family in land plants and are required for various post-transcriptional steps associated with RNA in plant organelles. The moss Physcomitrella patens PPR protein, PpPPR_38, is a nuclear-encoded chloroplast protein and was previously shown to be involved in the maturation step of chloroplast clpP pre-mRNA. To understand precisely the molecular function of PpPPR_38, we prepared recombinant PpPPR_38 protein and characterized it in maturation steps of clpP pre-mRNA. In vitro RNA-binding assays showed that the recombinant protein strongly bound to the clpP-5'-rps12 intergenic region, which is highly AU-rich and includes an inverted repeat sequence potentially forming a stem-loop structure. Digestion of the bound RNA region by RNase V1 was significantly accelerated by the addition of the recombinant protein. This strongly suggests that the binding of PpPPR_38 facilitates the formation of a stable stem-loop structure. An in vitro degradation assay using chloroplast lysates gave rise to the possibility that the stable stem-loop structure formed by PpPPR_38 contributes the correct intergenic RNA cleavage and protection of mature clpP mRNA against 3' to 5' exoribonuclease. Because an RNA-binding assay also showed weak binding to the clpP first exon-intron region, PpPPR_38 is likely to be related to the splicing of clpP pre-mRNA. Taking together all of the above findings, we conclude that PpPPR_38 is necessary for several steps in the clpP mRNA maturation process.