A Combinatorial Amino Acid Code for RNA Recognition by Pentatricopeptide Repeat Proteins

Abstract

The pentatricopeptide repeat (PPR) is a helical repeat motif found in an exceptionally large family of RNA–binding proteins that functions in mitochondrial and chloroplast gene expression. PPR proteins harbor between 2 and 30 repeats and typically bind single-stranded RNA in a sequence-specific fashion. However, the basis for sequence-specific RNA recognition by PPR tracts has been unknown. We used computational methods to infer a code for nucleotide recognition involving two amino acids in each repeat, and we validated this model by recoding a PPR protein to bind novel RNA sequences in vitro. Our results show that PPR tracts bind RNA via a modular recognition mechanism that differs from previously described RNA–protein recognition modes and that underpins a natural library of specific protein/RNA partners of unprecedented size and diversity. These findings provide a significant step toward the prediction of native binding sites of the enormous number of PPR proteins found in nature. Furthermore, the extraordinary evolutionary plasticity of the PPR family suggests that the PPR scaffold will be particularly amenable to redesign for new sequence specificities and functions.

An artificial PPR scaffold for programmable RNA recognition

“…The design of the final cPPR protein consisted of eight identical repeats because (i) it is of a manageable size for cloning and expression, (ii) based on previous experience working with pumilio and FBF homology (PUF) proteins this likely strikes a balance between effective binding and non-specific association 26 and (iii) it would be predicted to be able to bind a contiguous RNA 8 . In addition to the repeated consensus units, two extra features were added to the cPPR design: nucleating N-terminal cap residues (Met-Gly-AsnSer) and a C-terminal solvating helix.…”

“…The particular amino acids found at positions 4 and 34 of our cPPR are asparagine and glutamate, respectively ('ND'). Bioinformatic and recent structural analysis predicted that this combination would bind uracil [8][9][10] . We performed RNA electrophoretic shift assays with RNA homopolymers and observed specific binding of our designed cPPR to poly(U) RNA but no other RNA homopolymer (Fig.…”

“…Computational studies of a large family of RNA-binding proteins, known as pentatricopeptide repeat (PPR) proteins, have predicted that they bind their targets in a modular and sequencespecific manner [8][9][10] . PPR proteins contain a repeated motif that is typically 35 amino acids in length and folds into two anti-parallel alpha helices [11][12][13][14] .…”

“…Some PPR proteins appear to consist almost entirely of tandem PPRs, while some contain other domains, such as endonuclease or protein interaction domains [15][16][17] . Statistical correlations between specific PPR residues and RNA bases within their binding sites have elucidated a potential code for RNA recognition by PPR proteins [8][9][10] . A significant correlation was found between the identities of amino acids at positions 4 and 34 and particular bases within the RNA footprint [8][9][10] .…”

“…Statistical correlations between specific PPR residues and RNA bases within their binding sites have elucidated a potential code for RNA recognition by PPR proteins [8][9][10] . A significant correlation was found between the identities of amino acids at positions 4 and 34 and particular bases within the RNA footprint [8][9][10] . Furthermore, one study indicated that the identity of the amino acid at position 1 might fine-tune base recognition 9 .…”