A Conserved Lysine in an Ion-Pair with a Catalytic Glutamate Is Critical for U-to-C RNA Editing but Restricts C-to-U RNA Editing.

Plants make pyrimidine base substitutions in organellar mRNAs through the action of sequence-specific nuclear-encoded enzymes. Pentatricopeptide repeat (PPR) proteins are essential for ensuring specificity, while the enzymatic DYW domain is often present at the C-terminus of a PPR protein and dependent on the variant possessing C-to-U and/or U-to-C RNA editing activities. Expression of exogenous DYW-KP variant enzymes in bacteria leads to the modification of RNAs suggestive of U-to-C base changes.

Crosslinking of base-modified RNAs by synthetic DYW-KP base editors implicates an enzymatic lysine as the nitrogen donor for U-to-C RNA editing.

Sequence-specific cytidine to uridine (C-to-U) and adenosine to inosine editing tools can alter RNA and DNA sequences and utilize a hydrolytic deamination mechanism requiring an active site zinc ion and a glutamate residue. In plant organelles, DYW-PG domain containing enzymes catalyze C-to-U edits through the canonical deamination mechanism. Proteins developed from consensus sequences of the related DYW-KP domain family catalyze what initially appeared to be uridine to cytidine (U-to-C) edits leading to this investigation into the U-to-C editing mechanism.

RIP-Seq analysis of non-PPR chloroplast editing factors reveals broad RNA interactions and enrichment of less efficiently translated RNAs by OZ1 and ORRM1 complexes.

C-to-U RNA editing in angiosperm chloroplasts requires a large suite of proteins bound together in the editosome. The editosome is comprised of PPR proteins, RIP/MORFs, OZ proteins, and ORRM proteins that physically interact in high molecular weight complexes. The specific functions of non-PPR editing factors in the editosome are unclear, however, specific subsets of editing sites are affected by absence of non-PPR editing factors.

A ribonuclease activity linked to DYW1 in vitro is inhibited by RIP/MORF proteins.

Organellar C-to-U RNA editing in plants occurs in complexes composed of various classes of nuclear-encoded proteins. DYW-deaminases are zinc metalloenzymes that catalyze hydrolytic deamination required for C-to-U modification editing. Solved crystal structures for DYW-deaminase domains display all structural features consistent with a canonical cytidine deamination mechanism.

A RanBP2-type zinc finger protein functions in intron splicing in Arabidopsis mitochondria and is involved in the biogenesis of respiratory complex I.

The RanBP2 zinc finger (Znf) domain is a prevalent domain that mediates protein interaction and RNA binding. In Arabidopsis, a clade of four RanBP2 Znf-containing proteins, named the Organelle Zinc (OZ) finger family, are known or predicted to be targeted to either the mitochondria or the plastids. Previously we reported that OZ1 is absolutely required for the editing of 14 sites in chloroplasts.

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

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.

Stable native RIP9 complexes associate with C-to-U RNA editing activity, PPRs, RIPs, OZ1, ORRM1 and ISE2.

The mitochondrial and chloroplast mRNAs of the majority of land plants are modified through cytidine to uridine (C-to-U) RNA editing. Previously, forward and reverse genetic screens demonstrated a requirement for pentatricopeptide repeat (PPR) proteins for RNA editing. Moreover, chloroplast editing factors OZ1, RIP2, RIP9 and ORRM1 were identified in co-immunoprecipitation (co-IP) experiments, albeit the minimal complex sufficient for editing activity was never deduced.

A protein with an unusually short PPR domain, MEF8, affects editing at over 60 Arabidopsis mitochondrial C targets of RNA editing.

An RNA-seq approach was used to investigate the role of a PLS-subfamily pentatricopeptide repeat protein, Mitochondrial Editing Factor 8 (MEF8), on editing in Arabidopsis mitochondria and plastids. MEF8 has an intact DYW domain, but possesses an unusually short PLS repeat region of only five repeats. The MEF8 T-DNA insertion (mef8) line exhibited reduced editing at 38 mitochondrial editing sites and increased editing at 24 sites; therefore the absence of MEF8 affects 11% of the mitochondrial editome.

A conserved glutamate residue in the C-terminal deaminase domain of pentatricopeptide repeat proteins is required for RNA editing activity.

Many transcripts expressed from plant organelle genomes are modified by C-to-U RNA editing. Nuclear encoded pentatricopeptide repeat (PPR) proteins include an RNA binding domain that provides site specificity. In addition, many PPR proteins include a C-terminal DYW deaminase domain with characteristic zinc binding motifs (CXXC, HXE) and has recently been shown to bind zinc ions.

Identification of two pentatricopeptide repeat genes required for RNA editing and zinc binding by C-terminal cytidine deaminase-like domains.

Many transcripts expressed from plant organelle genomes are modified by C-to-U RNA editing. Nuclear encoded pentatricopeptide repeat (PPR) proteins are required as RNA binding specificity determinants in the RNA editing mechanism. Bioinformatic analysis has shown that most of the Arabidopsis PPR proteins necessary for RNA editing events include a C-terminal portion that shares structural characteristics with a superfamily of deaminases.

Monoclonal antibody heterogeneity analysis and deamidation monitoring with high-performance cation-exchange chromatofocusing using simple, two component buffer systems.

The use of either a polyampholyte buffer or a simple buffer system for the high-performance cation-exchange chromatofocusing of monoclonal antibodies is demonstrated for the case where the pH gradient is produced entirely inside the column and with no external mixing of buffers. The simple buffer system used was composed of two buffering species, one which becomes adsorbed onto the column packing and one which does not adsorb, together with an adsorbed ion that does not participate in acid-base equilibrium. The method which employs the simple buffer system is capable of producing a gradual pH gradient in the neutral to acidic pH range that can be adjusted by proper selection of the starting and ending pH values for the gradient as well as the buffering species concentration, pKa, and molecular size.

Molecular evolution of pentatricopeptide repeat genes reveals truncation in species lacking an editing target and structural domains under distinct selective pressures.

Background: Pentatricopeptide repeat (PPR) proteins are required for numerous RNA processing events in plant organelles including C-to-U editing, splicing, stabilization, and cleavage. Fifteen PPR proteins are known to be required for RNA editing at 21 sites in Arabidopsis chloroplasts, and belong to the PLS class of PPR proteins. In this study, we investigate the co-evolution of four PPR genes (CRR4, CRR21, CLB19, and OTP82) and their six editing targets in Brassicaceae species.

Pentatricopeptide repeat proteins constrain genome evolution in chloroplasts.

Higher plants encode hundreds of pentatricopeptide repeat proteins (PPRs) that are involved in several types of RNA processing reactions. Most PPR genes are predicted to be targeted to chloroplasts or mitochondria, and many are known to affect organellar gene expression. In some cases, RNA binding has been directly demonstrated, and the sequences of the cis-elements are known.

High conservation of a 5' element required for RNA editing of a C target in chloroplast psbE transcripts.

C-to-U editing modifies 30-40 distinct nucleotides within higher-plant chloroplast transcripts. Many C targets are located at the same position in homologous genes from different plants; these either could have emerged independently or could share a common origin. The 5' sequence GCCGUU, required for editing of C214 in tobacco psbE in vitro, is one of the few identified editing cis-elements.

Cross-competition in editing of chloroplast RNA transcripts in vitro implicates sharing of trans-factors between different C targets.

C-->U plant organellar RNA editing is required for the translation of evolutionarily conserved and functional proteins. 28 different C targets of RNA editing have been identified in maize chloroplasts, and hundreds of Cs are edited in mitochondria. Mutant analysis in Arabidopsis has indicated that absence of a single site-specific recognition protein can result in loss of editing of a single C target, raising the possibility that each C target requires a recognition protein.

Assay of editing of exogenous RNAs in chloroplast extracts of Arabidopsis, maize, pea, and tobacco.

Nucleotides within transcripts of chloroplasts and mitochondria are modified through C-to-U RNA editing in vascular plants. The specific protein components and enzymatic machinery required for editing have not been defined. A consensus sequence is not present around all editing sites, complicating the discovery of cis-sequence elements critical for editing.

Identification of a sequence motif critical for editing of a tobacco chloroplast transcript.

Nucleotides are specifically and efficiently targeted for modification from C to U within transcripts of chloroplasts in higher plants. Although the enzymatic apparatus responsible for altering C to U has not been identified, the sequences surrounding editing sites are known to contain information essential for efficient editing. We set out to determine the nucleotides that are critical for editing of a particular C, NTpsbE C214, in chloroplast transcripts in tobacco.

Sequence elements critical for efficient RNA editing of a tobacco chloroplast transcript in vivo and in vitro.

In tobacco chloroplast transcripts 34 nt are efficiently edited to U. No common consensus region is present around all editing sites; however, sites can be grouped in clusters that share short common sequences. Transgene transcripts carrying either the wild-type -31/+22 or -31/+60 sequence near NTrpoB C473, an editing site within tobacco rpoB transcripts, or three different mutated sequences, were all highly edited in vivo.

Substrate and cofactor requirements for RNA editing of chloroplast transcripts in Arabidopsis in vitro.

None of the macromolecular components of the chloroplast RNA editing apparatus has yet been identified. In order to facilitate biochemical purification and characterization of the chloroplast RNA editing apparatus, we have identified conditions suitable for production of chloroplast extracts from the model plant Arabidopsis that are capable of editing exogenous substrates produced by in vitro transcription. A simple poisoned primer extension assay readily quantified editing extent of mutated and wild-type substrates.