The pentatricopeptide repeat (PPR) is a helical repeat motif found in an exceptionally large family of RNA–binding proteins that functions in mitochondrial and chloroplast gene expression. PPR proteins harbor between 2 and 30 repeats and typically bind single-stranded RNA in a sequence-specific fashion. However, the basis for sequence-specific RNA recognition by PPR tracts has been unknown. We used computational methods to infer a code for nucleotide recognition involving two amino acids in each repeat, and we validated this model by recoding a PPR protein to bind novel RNA sequences in vitro. Our results show that PPR tracts bind RNA via a modular recognition mechanism that differs from previously described RNA–protein recognition modes and that underpins a natural library of specific protein/RNA partners of unprecedented size and diversity. These findings provide a significant step toward the prediction of native binding sites of the enormous number of PPR proteins found in nature. Furthermore, the extraordinary evolutionary plasticity of the PPR family suggests that the PPR scaffold will be particularly amenable to redesign for new sequence specificities and functions.

SummaryVirescence, a phenotype in which leaves green more slowly than usual, is recognized to play a role in protection from photo-oxidative damage before healthy chloroplasts are developed. The elucidation of the molecular mechanisms underlying virescence will provide insights into how the development of chloroplasts is controlled. In this study, we find that knockout alleles of Yellow Seedlings 1 (YS1) in Arabidopsis lead to a virescent phenotype, which disappears by 3 weeks after germination. The ys1 mutation resulted in marked decreases in photosynthetic capacity and photosynthetic pigment complexes, and disturbed ultrastructure of thylakoid membranes in 8-day-old seedlings. However, cotyledons of ys1 seedlings pre-treated in the dark for 5 days turn green almost as fast as the wild type in light, revealing that the developmental defects in ys1 are limited to the first few days after germination. Inspection of all known plastid RNA editing and splicing events revealed that YS1 is absolutely required for editing of site 25992 in rpoB transcripts encoding the beta subunit of the plastid-encoded RNA polymerase (PEP). YS1 is a nuclear-encoded chloroplast-localized pentatricopeptide repeat protein differing from previously described editing factors in that it has a C-terminal DYW motif. A defect in PEP activity is consistent with the changes in plastid transcript patterns observed in ys1 seedlings. We conclude that the activity of PEP containing RpoB translated from unedited transcripts is insufficient to support rapid chloroplast differentiation.

ORCID IDs: 0000-0003-0947-2648 (P.K.); 0000-0002-9584-6783 (C.S.B.); 0000-0001-5300-1216 (I.S.)RNA editing factors of the pentatricopeptide repeat (PPR) family show a very high degree of sequence specificity in the recognition of their target sites. A molecular basis for target recognition by editing factors has been proposed based on statistical correlations but has not been tested experimentally. To achieve this, we systematically mutated the pentatricopeptide motifs in the Arabidopsis thaliana RNA editing factor CLB19 to investigate their individual contribution to RNA recognition. We find that the motifs contributing significantly to the specificity of binding follow the previously proposed recognition rules, distinguishing primarily between purines and pyrimidines. Our results are consistent with proposals that each motif recognizes one nucleotide in the RNA target with the protein aligned parallel to the RNA and contiguous motifs aligned with contiguous nucleotides such that the final PPR motif aligns four nucleotides upstream of the edited cytidine. By altering S motifs in CLB19 and another editing factor, OTP82, and using the modified proteins to attempt to complement the respective mutants, we demonstrate that we can predictably alter the specificity of these factors in vivo.