Crystal structures of the Arabidopsis thaliana organellar RNA editing factors MORF1 and MORF9.

In flowering plant plastids and mitochondria, multiple organellar RNA editing factor (MORF/RIP) proteins are required at most sites for efficient C to U RNA editing catalyzed by the RNA editosome. MORF proteins harbor a conserved stretch of residues (MORF-box), form homo- and heteromers and interact with selected PPR (pentatricopeptide repeat) proteins, which recognize each editing site. The molecular function of the MORF-box remains elusive since it shares no sequence similarity with known domains. We determined structures of the A. thaliana mitochondrial MORF1 and chloroplast MORF9 MORF-boxes which both adopt a novel globular fold (MORF domain). Our structures state a paradigmatic model for MORF domains and their specific dimerization via a hydrophobic interface. We cross-validate the interface by yeast two-hybrid studies and pulldown assays employing structure-based mutants. We find a structural similarity of the MORF domain to an N-terminal ferredoxin-like domain (NFLD), which confers RNA substrate positioning in bacterial 4-thio-uracil tRNA synthetases, implying direct RNA contacts of MORF proteins during RNA editing. With the MORF1 and MORF9 structures we elucidate a yet unknown fold, corroborate MORF interaction studies, validate the mechanism of MORF multimerization by structure-based mutants and pave the way towards a complete structural characterization of the plant RNA editosome.

DYW domain structures imply an unusual regulation principle in plant organellar RNA editing catalysis.

RNA editosomes selectively deaminate cytidines to uridines in plant organellar transcripts-mostly to restore protein functionality and consequently facilitate mitochondrial and chloroplast function. The RNA editosomal pentatricopeptide repeat proteins serve target RNA recognition, whereas the intensively studied DYW domain elicits catalysis. Here we present structures and functional data of a DYW domain in an inactive ground state and activated. DYW domains harbour a cytidine deaminase fold and a C-terminal DYW motif, with catalytic and structural zinc atoms, respectively. A conserved gating domain within the deaminase fold regulates the active site sterically and mechanistically in a process that we termed gated zinc shutter. Based on the structures, an autoinhibited ground state and its activation are cross-validated by RNA editing assays and differential scanning fluorimetry. We anticipate that, in vivo, the framework of an active plant RNA editosome triggers the release of DYW autoinhibition to ensure a controlled and coordinated cytidine deamination playing a key role in mitochondrial and chloroplast homeostasis.

RNA editing mutants as surrogates for mitochondrial SNP mutants.

In terrestrial plants, RNA editing converts specific cytidines to uridines in mitochondrial and plastidic transcripts. Most of these events appear to be important for proper function of organellar encoded genes, since translated proteins from edited mRNAs show higher similarity with evolutionary conserved polypeptide sequences. So far about 100 nuclear encoded proteins have been characterized as RNA editing factors in plant organelles. Respective RNA editing mutants reduce or lose editing activity at different sites and display various macroscopic phenotypes from pale or albino in the case of chloroplasts to growth retardation or even embryonic lethality. Therefore, RNA editing mutants can be a useful resource of surrogate mutants for organellar encoded genes, especially for mitochondrially encoded genes that it is so far unfeasible to manipulate. However, connections between RNA editing defects and observed phenotypes in the mutants are often hard to elucidate, since RNA editing factors often target multiple RNA sites in different genes simultaneously. In this review article, we summarize the physiological aspects of respective RNA editing mutants and discuss them as surrogate mutants for functional analysis of mitochondrially encoded genes.

The conserved domain in MORF proteins has distinct affinities to the PPR and E elements in PPR RNA editing factors.

In plant organelles specific nucleotide motifs at C to U RNA editing sites are recognized by the PLS-class of pentatricopeptide repeat (PPR) proteins, which are additionally characterized by a C-terminal E domain. The PPR elements bind the nucleotides in the target RNA, while the function of the E domain has remained unknown. At most sites RNA editing also requires multiple organellar RNA editing factor (MORF) proteins. To understand how these two types of proteins are involved in RNA editing complexes, we systematically analyzed their protein-protein interactions. In vivo pull-down and yeast two-hybrid assays show that MORF proteins connect with selected PPR proteins. In a loss of function mutant of MORF1, a single amino acid alteration in the conserved MORF domain abrogates interactions with many PLS-class PPR proteins, implying the requirement of direct interaction to PPR proteins for the RNA editing function of MORF1. Subfragment analyses show that predominantly the N-terminal/central regions of the MORF domain in MORF1 and MORF3 bind the PPR proteins. Within the PPR proteins, the E domains in addition to PPR elements contact MORF proteins. In chimeric PPR proteins, different E domains alter the specificity of the interaction with MORF proteins. The selective interactions between E domain containing PPR and MORF proteins suggest that the E domains and MORF proteins play a key role for specific protein complexes to assemble at different RNA editing sites.