Ribonucleoprotein Capture by in Vivo Expression of a Designer Pentatricopeptide Repeat Protein in Arabidopsis

Pentatricopeptide repeat (PPR) proteins bind RNA via a mechanism that facilitates the customization of sequence specificity. However, natural PPR proteins have irregular features that limit the degree to which their specificity can be predicted and customized. We demonstrate here that artificial PPR proteins built from consensus PPR motifs selectively bind the intended RNA in vivo, and we use this property to develop a new tool for ribonucleoprotein characterization. We show by RNA coimmunoprecipitation sequencing (RIP-seq) that artificial PPR proteins designed to bind the Arabidopsis ( Arabidopsis thaliana ) chloroplast psbA mRNA bind with high specificity to psbA mRNA in vivo. Analysis of coimmunoprecipitating proteins by mass spectrometry showed the psbA translational activator HCF173 and two RNA binding proteins of unknown function (CP33C and SRRP1) to be highly enriched. RIP-seq revealed that these proteins are bound primarily to psbA RNA in vivo, and precise mapping of the HCF173 and CP33C binding sites placed them in different locations on psbA mRNA. These results demonstrate that artificial PPR proteins can be tailored to bind specific endogenous RNAs in vivo, add to the toolkit for characterizing native ribonucleoproteins, and open the door to other applications that rely on the ability to target a protein to a specified RNA sequence.