Structural basis for the modular recognition of single-stranded RNA by PPR proteins

Abstract

Pentatricopeptide repeat (PPR) proteins represent a large family of sequence-specific RNA-binding proteins that are involved in multiple aspects of RNA metabolism. PPR proteins, which are found in exceptionally large numbers in the mitochondria and chloroplasts of terrestrial plants, recognize single-stranded RNA (ssRNA) in a modular fashion. The maize chloroplast protein PPR10 binds to two similar RNA sequences from the ATPI-ATPH and PSAJ-RPL33 intergenic regions, referred to as ATPH and PSAJ, respectively. By protecting the target RNA elements from 5' or 3' exonucleases, PPR10 defines the corresponding 5' and 3' messenger RNA termini. Despite rigorous functional characterizations, the structural basis of sequence-specific ssRNA recognition by PPR proteins remains to be elucidated. Here we report the crystal structures of PPR10 in RNA-free and RNA-bound states at resolutions of 2.85 and 2.45 Å, respectively. In the absence of RNA binding, the nineteen repeats of PPR10 are assembled into a right-handed superhelical spiral. PPR10 forms an antiparallel, intertwined homodimer and exhibits considerable conformational changes upon binding to its target ssRNA, an 18-nucleotide PSAJ element. Six nucleotides of PSAJ are specifically recognized by six corresponding PPR10 repeats following the predicted code. The molecular basis for the specific and modular recognition of RNA bases A, G and U is revealed. The structural elucidation of RNA recognition by PPR proteins provides an important framework for potential biotechnological applications of PPR proteins in RNA-related research areas.

An artificial PPR scaffold for programmable RNA recognition

“…All together, the presented data demonstrate that repeats 2, 3, 4 and 5 from our cPPR recognize individual nucleotides and strongly suggest that all repeats within the cPPR are contributing to RNA recognition. These results biochemically validate the computationally predicted code for base recognition as well as previous structural observations 18 , and demonstrate that our engineered cPPRs can bind RNAs in a programmable manner.…”

“…A previous study of PPR10 observed direct hydrogen bonding of adenine, guanine and uracil by S, T and N residues, respectively, at position 4 of the corresponding PPR modules 18 . Specific recognition of cytosine has not previously been observed, although direct hydrogen bonding by N4 was predicted 18 . In our structures, the electrostatic properties highlight the proposed nucleic acid binding groove in the inner face of the superhelix (Fig.…”

“…5b-d). Because the recognition of cytosine by a PPR repeat was not observed in the PPR10-PSAJ RNA structure 18 , or any other structure to date, we modelled cytosine recognition based on uridine. Despite the absence of direct hydrogen bonds between the hydroxyl group and the D34 position of PPR10 or our cPPR, uridine is recognized specifically.…”

“…Furthermore, one study indicated that the identity of the amino acid at position 1 might fine-tune base recognition 9 . Recently the structures of two distinct PPR-RNA complexes have been described at atomic resolution 18,19 . Surprisingly, although some elements of a modular recognition code were observed, the majority of the RNA chains in both of these structures were bound independently of the sequence of their RNA bases.…”

“…For example, extensive mutagenesis and truncations of the PPR10 protein were required for production of soluble protein for crystallization studies 18 . This problem has severely delayed the elucidation of the mechanisms of PPR-RNA binding and their potential use as tools.…”