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

Michael L Hayes,Paola I Santibanez

doi:10.1074/jbc.ra119.011790

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.

Highlights

Pentatricopeptide repeat (PPR) proteins with C-terminal DYW domains are present in organisms that undergo C-to-U editing of organelle RNA transcripts
Similar transition state analogs have been shown to reduce activity for Escherichia coli cytidine deaminase [2], adenosine deaminase acting on RNA (ADAR)2 [26], and Apolipoprotein B mRNA Editing Enzyme Catalytic Subunit 3G (APOBEC3G) [27]
2 The abbreviations used are: ADAR, adenosine deaminase acting on RNA; PPR, pentatricopeptide repeat; PHEN, 1–10-o-phenanthroline; ATP␥S, adenosine 5Ј-O-(3-thiotriphosphate); AMP-PCP, ␤,␥-methyleneadenosine 5Ј-triphosphate; AMP-CP, ␣,␤-methyleneadenosine 5Ј-diphosphate; THU, tetrahydrouridine; Cy5, indodicarbocyanine; ZEB, zebuarine; AZA, 5-azacytdine; PPE, poisoned primer extension; ESI, electrospray ionization; Inductively coupled plasma (ICP)-OES, inductively coupled plasma-optical emission spectrometry; APOBEC3G, apolipoprotein B mRNA-editing enzyme catalytic subunit 3G; MBP, maltose-binding protein; MALS, multiangle light scattering

Summary

ARTICLE cro

A plant pentatricopeptide repeat protein with a DYWdeaminase domain is sufficient for catalyzing C-to-U RNA editing in vitro. 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. RNA oligonucleotides with a single incorporated zebularine 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. The C terminus lacks one of the nucleotide-interacting loops proposed for activation-induced cytidine deaminase [23], possesses a pair of unique conserved cysteines that likely coordinate an additional zinc residue [9], and ends in the C-terminal DYW trio of amino acids residues with unknown roles. We provide evidence that RNA editing might be regulated by the adenylate energy charge of the organelle through allostery and is not inhibited by the addition of putative transition state analogs zebularine hydrate and tetrahydrouridine

Results

Discussion

Zinc from protein

Expression of PPR proteins

MALS analysis

Zinc metal analysis