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RNA-binding specificity landscapes of designer pentatricopeptide repeat proteins elucidate principles of PPR-RNA interactions.

ABSTRACT: Pentatricopeptide repeat (PPR) proteins are helical-repeat proteins that offer a promising scaffold for the engineering of proteins to bind specified RNAs. PPR tracts bind RNA in a modular 1-repeat, 1-nucleotide fashion. An amino acid code specifying the bound nucleotide has been elucidated. However, this code does not fully explain the sequence specificity of native PPR proteins. Furthermore, it does not address nuances such as the contribution toward binding affinity of various repeat-nucleotide pairs or the impact of mismatches between a repeat and aligning nucleotide. We used an in vitro bind-n-seq approach to describe the population of sequences bound by four artificial PPR proteins built from consensus scaffolds. The specificity of these proteins can be accounted for by canonical code-based nucleotide recognition. The results show, however, that interactions near the 3'-end of binding sites make less contribution to binding affinity than do those near the 5'-end, that proteins with 11 and 14 repeats exhibit similar affinity for their intended targets but 14-repeats are more permissive for mismatches, and that purine-binding repeats are less tolerant of transversion mismatches than are pyrimidine-binding motifs. These findings have implications for mechanisms that establish PPR-RNA interactions and for optimizing PPR design to minimize off-target interactions.

SUBMITTER: Miranda RG

PROVIDER: S-EPMC5861457 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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RNA-binding specificity landscapes of designer pentatricopeptide repeat proteins elucidate principles of PPR-RNA interactions.

Miranda Rafael G RG McDermott James J JJ Barkan Alice A

Nucleic acids research 20180301 5

Pentatricopeptide repeat (PPR) proteins are helical-repeat proteins that offer a promising scaffold for the engineering of proteins to bind specified RNAs. PPR tracts bind RNA in a modular 1-repeat, 1-nucleotide fashion. An amino acid code specifying the bound nucleotide has been elucidated. However, this code does not fully explain the sequence specificity of native PPR proteins. Furthermore, it does not address nuances such as the contribution toward binding affinity of various repeat-nucleoti ...[more]

PMID: 29294070

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Project description:BackgroundThe Campanuloideae (Campanulaceae) are a highly diverse clade of angiosperms found mostly in the Northern Hemisphere, with the highest diversity in temperate areas of the Old World. Chloroplast markers have greatly improved our understanding of this clade but many relationships remain unclear primarily due to low levels of molecular evolution and recent and rapid divergence. Furthermore, focusing solely on maternally inherited markers such as those from the chloroplast genome may obscure processes such as hybridization. In this study we explore the phylogenetic utility of two low-copy nuclear loci from the pentatricopeptide repeat gene family (PPR). Rapidly evolving nuclear loci may provide increased phylogenetic resolution in clades containing recently diverged or closely related taxa. We present results based on both chloroplast and low-copy nuclear loci and discuss the utility of such markers to resolve evolutionary relationships and infer hybridization events within the Campanuloideae clade.ResultsThe inclusion of low-copy nuclear genes into the analyses provides increased phylogenetic resolution in two species-rich clades containing recently diverged taxa. We also obtain support for the placement of two early diverging lineages (Jasione and Musschia-Gadellia clades) that have previously been unresolved. Furthermore, phylogenetic analyses of PPR loci revealed potential hybridization events for a number of taxa (e.g., Campanula pelviformis and Legousia species). These loci offer greater overall topological support than obtained with plastid DNA alone.ConclusionThis study represents the first inclusion of low-copy nuclear genes for phylogenetic reconstruction in Campanuloideae. The two PPR loci were easy to sequence, required no cloning, and the sequence alignments were straightforward across the entire Campanuloideae clade. Although potentially complicated by incomplete lineage sorting, these markers proved useful for understanding the processes of reticulate evolution and resolving relationships at a wide range of phylogenetic levels. Our results stress the importance of including multiple, independent loci in phylogenetic analyses.

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RNA-binding specificity landscapes of designer pentatricopeptide repeat proteins elucidate principles of PPR-RNA interactions.

Publications

RNA-binding specificity landscapes of designer pentatricopeptide repeat proteins elucidate principles of PPR-RNA interactions.

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