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Plant nuclear genomes encode hundreds of predicted organellar RNA binding proteins, few of which have been connected with their physiological RNA substrates and functions. In fact, among the largest family of putative RNA binding proteins in plants, the pentatricopeptide repeat (PPR) family, no physiologically relevant RNA ligands have been firmly established. We used the chloroplast-splicing factor CAF1 to demonstrate the fidelity of a microarray-based method for identifying RNAs associated with specific proteins in chloroplast extract. We then used the same method to identify RNAs associated with the maize (Zea mays) PPR protein CRP1. Two mRNAs whose translation is CRP1-dependent were strongly and specifically enriched in CRP1 coimmunoprecipitations. These interactions establish CRP1 as a translational regulator by showing that the translation defects in crp1 mutants are a direct consequence of the absence of CRP1. Additional experiments localized these interactions to the 59 untranslated regions and suggested a possible CRP1 interaction motif. These results enhance understanding of the PPR protein family by showing that a PPR protein influences gene expression through association with specific mRNAs in vivo, suggesting an unusual mode of RNA binding for PPR proteins, and highlighting the possibility that translational regulation may be a particularly common function of PPR proteins. Analogous methods should have broad application for the study of native RNA-protein interactions in both mitochondria and chloroplasts.

The pentatricopeptide repeat (PPR) is a degenerate 35-amino acid repeat motif that is widely distributed among eukaryotes. Genetic, biochemical, and bioinformatic data suggest that many PPR proteins influence specific posttranscriptional steps in mitochondrial or chloroplast gene expression and that they may typically bind RNA. However, biological functions have been determined for only a few PPR proteins, and with few exceptions, substrate RNAs are unknown. To gain insight into the functions and substrates of the PPR protein family, we characterized the maize (Zea mays) nuclear gene ppr4, which encodes a chloroplast-targeted protein harboring both a PPR tract and an RNA recognition motif. Microarray analysis of RNA that coimmunoprecipitates with PPR4 showed that PPR4 is associated in vivo with the first intron of the plastid rps12 pre-mRNA, a group II intron that is transcribed in segments and spliced in trans. ppr4 mutants were recovered through a reverse-genetic screen and shown to be defective for rps12 trans-splicing. The observations that PPR4 is associated in vivo with rps12-intron 1 and that it is also required for its splicing demonstrate that PPR4 is an rps12 trans-splicing factor. These findings add trans-splicing to the list of RNA-related functions associated with PPR proteins and suggest that plastid group II trans-splicing is performed by different machineries in vascular plants and algae.