PCIS1, encoded by a pentatricopeptide protein co-expressed gene, is required for splicing of three mitochondrial nad transcripts in angiosperms.

Group II introns are large catalytic RNAs, which reside mainly within genes encoding respiratory complex I (CI) subunits in angiosperms' mitochondria. Genetic and biochemical analyses led to the identification of many nuclear-encoded factors that facilitate the splicing of the degenerated organellar introns in plants. Here, we describe the analysis of the PPR Co-expressed Intron Splicing1 (PCIS1) factor, which was identified in-silico by its co-expression pattern with many PPR proteins. PCIS1 is well conserved in land plants but has no sequence similarity with any known protein motifs. PCIS1 mutant lines are arrested in embryogenesis and can be maintained by the temporal expression of the gene under the embryo-specific ABI3 promoter. The pABI3::PCIS1 mutant plants display low germination and stunted growth phenotypes. RNA-seq and RT-qPCR analyses of wild type and mutant plants indicated that PCIS1 is a novel splicing cofactor that is pivotal for the maturation of several nad transcripts in Arabidopsis mitochondria. These phenotypes are tightly associated with respiratory complex I defects and altered plant growth. Our data further emphasizes the key roles of nuclear-encoded cofactors that regulate the maturation and expression of mitochondrial transcripts for the biogenesis of the oxidative phosphorylation (OXPHOS) system, and hence for plant physiology. The discovery of novel splicing factors other than typical RNA-binding proteins suggests further complexity of splicing mechanisms in plant mitochondria.

DYW deaminase domain has a distinct preference for neighboring nucleotides of the target RNA editing sites.

C-to-U RNA editing sites in plant organelles show a strong bias for neighboring nucleotides. The nucleotide upstream of the target cytidine is typically C or U, whereas A and G are less common and rare, respectively. In pentatricopeptide repeat (PPR)-type RNA editing factors, the PPR domain specifically binds to the 5' sequence of target cytidines, whereas the DYW domain catalyzes the C-to-U deamination. We comprehensively analyzed the effects of neighboring nucleotides of the target cytidines using an Escherichia coli orthogonal system. Physcomitrium PPR56 efficiently edited target cytidines when the nucleotide upstream was U or C, whereas it barely edited when the position was G or the nucleotide downstream was C. This preference pattern, which corresponds well with the observed nucleotide bias for neighboring nucleotides in plant organelles, was altered when the DYW domain of OTP86 or DYW1 was adopted. The PPR56 chimeric proteins edited the target sites even when the -1 position was G. Our results suggest that the DYW domain possesses a distinct preference for the neighboring nucleotides of the target sites, thus contributing to target selection in addition to the existing selection determined by the PPR domain.

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.