An artificial PPR scaffold for programmable RNA recognition

Abstract

Pentatricopeptide repeat (PPR) proteins control diverse aspects of RNA metabolism in eukaryotic cells. Although recent computational and structural studies have provided insights into RNA recognition by PPR proteins, their highly insoluble nature and inconsistencies between predicted and observed modes of RNA binding have restricted our understanding of their biological functions and their use as tools. Here we use a consensus design strategy to create artificial PPR domains that are structurally robust and can be programmed for sequence-specific RNA binding. The atomic structures of these artificial PPR domains elucidate the structural basis for their stability and modelling of RNA-protein interactions provides mechanistic insights into the importance of RNA-binding residues and suggests modes of PPR-RNA association. The modular mode of RNA binding by PPR proteins holds great promise for the engineering of new tools to target RNA and to understand the mechanisms of gene regulation by natural PPR proteins.

The MTL1 Pentatricopeptide Repeat Protein Is Required for Both Translation and Splicing of the Mitochondrial NADH DEHYDROGENASE SUBUNIT7 mRNA in Arabidopsis

“…P-type PPR proteins were shown to participate in various aspects of organellar RNA processing, whereas PLS PPR proteins have been almost exclusively associated with C-to-U RNA editing (for review, see Barkan and Small, 2014;Hammani and Giegé, 2014). Recent crystal structures showed that PPR motifs adopt an antiparallel helixturn-helix fold whose repetition forms a solenoid-like structure (Ringel et al, 2011;Howard et al, 2012;Ban et al, 2013;Yin et al, 2013;Coquille et al, 2014;Gully et al, 2015). PPR tracks organize highly specific interaction domains that were shown to associate with single-stranded RNAs (Schmitz-Linneweber et al, 2005;Beick et al, 2008;Uyttewaal et al, 2008a;WilliamsCarrier et al, 2008;Pfalz et al, 2009;Cai et al, 2011;Hammani et al, 2011;Prikryl et al, 2011;Khrouchtchova et al, 2012;Manavski et al, 2012;Zhelyazkova et al, 2012;Ke et al, 2013;Yin et al, 2013).…”

Programmable RNA-binding protein composed of repeats of a single modular unit

“…Ideally one could target unmodified RNA, both for simplicity and to preserve as much native RNA structure and function as possible (4,5). It has been proposed that proteins such as the Caenorhabditis elegans Puf (6), the human PumHD (Pumilio homology domain) (7), or members of the pentatricopeptide family (8) could serve such a purpose. Each of these proteins is made of many similar units, each of which targets one RNA base.…”

PPR-SMR protein SOT1 has RNA endonuclease activity

“…The 2nd, 5th, and 35th (or 1st, 4th, and 34th or 3rd, 6th, and 1st in other numbering systems) residues at each repeat are considered to be RNA selection "codes" (9)(10)(11). Based on these codes, several PPR proteins have been successfully modified to recognize predictable RNA targets (9,(12)(13)(14)(15)(16)(17).…”

“…The 2nd, 5th, and 35th (or 1st, 4th, and 34th or 3rd, 6th, and 1st in other numbering systems) residues at each repeat are considered to be RNA selection "codes" (9-11). Based on these codes, several PPR proteins have been successfully modified to recognize predictable RNA targets (9,(12)(13)(14)(15)(16)(17).The small MutS-related (SMR) domain was originally identified at the C terminus of MutS2 in the cyanobacterium Synechocystis (18). SMR proteins are widely distributed in almost all organisms (19).…”