A plant pentatricopeptide repeat protein with a DYW-deaminase domain is sufficient for catalyzing C-to-U RNA editing in vitro

Abstract

Pentatricopeptide repeat (PPR) proteins with C-terminal DYW domains are present in organisms that undergo C-to-U editing of organelle RNA transcripts. PPR domains act as specificity factors through electrostatic interactions between a pair of polar residues and the nitrogenous bases of an RNA target. DYW-deaminase domains act as the editing enzyme. Two moss ( Physcomitrella patens ) PPR proteins containing DYW-deaminase domains, PPR65 and PPR56, can convert Cs to Us in cognate, exogenous RNA targets co-expressed in Escherichia coli . We show here that purified, recombinant PPR65 exhibits robust editase activity on synthetic RNAs containing cognate, mitochondrial PpccmFC sequences in vitro , indicating that a PPR protein with a DYW domain is solely sufficient for catalyzing C-to-U RNA editing in vitro . Monomeric fractions possessed the highest conversion efficiency, and oligomeric fractions had reduced activity. Inductively coupled plasma (ICP)–MS analysis indicated a stoichiometry of two zinc ions per highly active PPR65 monomer. Editing activity was sensitive to addition of zinc acetate or the zinc chelators 1,10- o -phenanthroline and EDTA. Addition of ATP or nonhydrolyzable nucleotide analogs stimulated PPR65-catalyzed RNA-editing activity on PpccmFC substrates, indicating potential allosteric regulation of PPR65 by ATP. Unlike for bacterial cytidine deaminase, addition of two putative transition-state analogs, zebularine and tetrahydrouridine, failed to disrupt RNA-editing activity. RNA oligonucleotides with a single incorporated zebularine also did not disrupt editing in vitro , suggesting that PPR65 cannot bind modified bases due to differences in the structure of the active site compared with other zinc-dependent nucleotide deaminases.