title: RNA-binding specificity landscape of the pentatricopeptide repeat protein PPR10.
abstract: Pentatricopeptide repeat (PPR) proteins comprise a large family of helical repeat proteins that influence gene expression in mitochondria and chloroplasts. PPR tracts can bind RNA via a modular one repeat-one nucleotide mechanism in which the nucleotide is specified by the identities of several amino acids in each repeat. This mode of recognition, the so-called PPR code, offers opportunities for the prediction of native PPR binding sites and the design of proteins to bind specified RNAs.However, a deep understanding of the parameters that dictate the affinity and specificity of PPR-RNA interactions is necessary to realize these goals. We report a comprehensive analysis of the sequence specificity of PPR10, a protein that binds similar RNA sequences of ∼18 nucleotides (nt) near the chloroplastatpHandpsaJgenes in maize. We assessed the contribution of each nucleotide in theatpHbinding site to PPR10 affinity in vitro by analyzing the effects of single-nucleotide changes at each position. In a complementary approach, the RNAs bound by PPR10 from partially randomized RNA pools were analyzed by deep sequencing. The results revealed three patches in which nucleotide identity has a major impact on binding affinity. These include 5 nt for which protein contacts were not observed in a PPR10-RNA crystal structure and 4 nt that are not explained by current views of the PPR code. These findings highlight aspects of PPR-RNA interactions that pose challenges for binding site prediction and design.
authors.affiliation: Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403, USA.
authors.forename: Rafael G
authors.initials: RG
authors.lastname: Miranda
authors.affiliation: Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403, USA.
authors.forename: Margarita
authors.initials: M
authors.lastname: Rojas
authors.affiliation: Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403, USA.
authors.forename: Michael P
authors.initials: MP
authors.lastname: Montgomery
authors.affiliation: Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403, USA.
authors.forename: Kyle P
authors.initials: KP
authors.lastname: Gribbin
authors.affiliation: Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403, USA abarkan@uoregon.edu.
authors.forename: Alice
authors.initials: A
authors.lastname: Barkan
mesh_terms: D001665:Binding Sites; D060365:Chloroplast Proteins / Q000235:genetics* / Q000378:metabolism; D002736:Chloroplasts / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D059372:Nucleotide Motifs; D045331:Photosystem I Protein Complex / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D018749:RNA, Plant / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D003313:Zea mays / Q000235:genetics* / Q000378:metabolism
chemical_list: D060365:Chloroplast Proteins; D045331:Photosystem I Protein Complex; D018749:RNA, Plant; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins
keywords: RNA-binding protein; bind-n-seq; chloroplast; helical repeat protein