An in vitro RNA editing system from cauliflower mitochondria: editing site recognition parameters can vary in different plant species

Most of the 400 RNA editing sites in flowering plant mitochondria are found in mRNAs. Consequently, the sequence vicinities of homologous sites are highly conserved between different species and are presumably recognized by likewise conserved trans-factors. To investigate the evolutionary adaptation to sequence variation, we have now analyzed the recognition elements of an editing site with divergent upstream sequences in the two species pea and cauliflower. This variation is tolerated at the site selected, because the upstream cis-elements reach into the 5'-UTR of the mRNA. To compare cis-recognition features in pea and cauliflower mitochondria, we developed a new in vitro RNA editing system for cauliflower. In vitro editing assays with deleted and mutated template RNAs show that the major recognition elements for both species are located within the conserved sequence. In cauliflower, however, the essential upstream nucleotides extend further upstream than they do in pea. In-depth analysis of single-nucleotide mutations reveals critical spacing of the editing site and the specific recognition elements, and shows that the +1 nucleotide identity is important in cauliflower, but not in pea.     

RNA editing in plant mitochondria

RNA editing in plant mitochondria alters nearly all mRNAs by C to U and U to C transitions. In some species more than 400 edited sites have been identified with significant effects on the encoded proteins. RNA editing occurs in higher and lower plants and presumably has evolved before the differentiation of land plants. Current research focuses on the elucidation of the biochemistry and the specificity determinants of RNA editing in plant mitochrondria.     

Trypanosomatids: mitochondrial RNA editing

RNA editing is a genetic regulatory process that was recently discovered in the mitochondrion of trypanosomatid parasites. It alters mRNA by the addition and deletion of uridines. Much remains to be learned about this process, including identification and characterization of the macromolecules that catalyze and regulate this process and the mechanism of editing.     

In vitro RNA editing in plant mitochondria does not require added energy

RNA editing in flowering plant mitochondria is investigated by in vitro assays. These cauliflower mitochondrial lysates require added NTP or dNTP. We have now resolved the reason for this requirement to be the inhibition of the RNA binding activity of the glutamate dehydrogenases (GDH). Both GDH1 and GDH2 were identified in RNA-protein cross-links. The inhibition of in vitro RNA editing by GDH is confirmed by the ability of the GDH-specific herbicide phosphinothricin to substitute for NTP. NADH and NADPH, but not NAD or NADP, can also replace NTP, suggesting that the NAD(P)H-binding-pocket configuration of the GDH contacts the RNA. RNA editing in plant mitochondria is thus intrinsically independent of added energy in the form of NTP.     

RNA editing in mitochondrial mRNA of trypanosomatids.

The editing of mRNA coding sequences by the modification, removal or addition of nucleotides has recently been recognized as another form of RNA processing. Studies of the extensive editing of mitochondrial mRNAs in trypanosomatids have revealed the involvement of small guide RNAs (gRNAs) which are encoded by the minicircles of kinetoplast DNA.     

RNA bulges as architectural and recognition motifs

RNA bulges constitute versatile structural motifs in the assembly of RNA architectures. Three-dimensional structures of RNA molecules and their complexes reveal the role of bulges in RNA architectures and illustrate the molecular mechanisms by which they confer intramolecular interactions and intermolecular recognition.     

Dynamic landscape of mitochondrial Cytidine-to-Uridine RNA editing in tobacco (Nicotiana tabacum) shows its tissue specificity

Plant Cell, Tissue and Organ Culture (PCTOC) - RNA editing is a prevalent nucleotide modification at the RNA level in higher plants. However, little is known about the dynamic distribution of RNA...     

The process of RNA editing in plant mitochondria

RNA editing changes more than 400 cytidines to uridines in the mRNAs of mitochondria in flowering plants. In other plants such as ferns and mosses, RNA editing reactions changing C to U and U to C are observed at almost equal frequencies. Development of transfection systems with isolated mitochondria and of in vitro systems with extracts from mitochondria has considerably improved our understanding of the recognition of specific editing sites in the last few years. These assays have also yielded information about the biochemical parameters, but the enzymes involved have not yet been identified. Here we summarize our present understanding of the process of RNA editing in flowering plant mitochondria.     

Computational analysis of RNA editing sites in plant mitochondrial genomes reveals similar information content and a sporadic distribution of editing sites

A computational analysis of RNA editing sites was performedon protein-coding sequences of plant mitochondrial genomes fromArabidopsis thaliana, Beta vulgaris, Brassica napus, and Oryzasativa. The distribution of nucleotides around edited and uneditedcytidines was compared in 41 nucleotide segments and included1481 edited cytidines and 21,390 unedited cytidines in the 4genomes. The distribution of nucleotides was examined in 1,2, and 3 nucleotide windows by comparison of nucleotide frequencyratios and relative entropy. The relative entropy analyses indicatethat information is encoded in the nucleotide sequences in the5 prime flank (–18 to –14, –13 to –10,–6 to –4, –2/–1) and the immediate 3prime flanking nucleotide (+1), and these regions may be importantin editing site recognition. The relative entropy was largewhen 2 or 3 nucleotide windows were analyzed, suggesting thatseveral contiguous nucleotides may be involved in editing siterecognition. RNA editing sites were frequently preceded by 2pyrimidines or AU and followed by a guanidine (HYCG) in themonocot and dicot mitochondrial genomes, and rarely precededby 2 purines. Analysis of chloroplast editing sites from a dicot,Nicotiana tabacum, and a monocot, Zea mays, revealed a similardistribution of nucleotides around editing sites (HYCA). Thesimilarity of this motif around editing sites in monocots anddicots in both mitochondria and chloroplasts suggests that amechanistic basis for this motif exists that is common in thesedifferent organelle and phylogenetic systems. The preferredsequence distribution around RNA editing sites may have an importantimpact on the acquisition of editing sites in evolution becausethe immediate sequence context of a cytidine residue may rendera cytidine editable or uneditable, and consequently determinewhether a T to C mutation at a specific position may be correctedby RNA editing. The distribution of editing sites in many protein-codingsequences is shown to be non-random with editing sites clusteredin groups separated by regions with no editing sites. The sporadicdistribution of editing sites could result from a mechanismof editing site loss by gene conversion utilizing edited sequenceinformation, possibly through an edited cDNA intermediate.     

Diversity and evolution of mitochondrial RNA editing systems

'RNA editing' describes the programmed alteration of the nucleotide sequence of an RNA species, relative to the sequence of the encoding DNA. The phenomenon encompasses two generic patterns of nucleotide change, 'insertion/deletion' and 'substitution', defined on the basis of whether the sequence of the edited RNA is colinear with the DNA sequence that encodes it. RNA editing is mediated by a variety of pathways that are mechanistically and evolutionarily unrelated. Messenger, ribosomal, transfer and viral RNAs all undergo editing in different systems, but well-documented cases of this phenomenon have so far been described only in eukaryotes, and most often in mitochondria. Editing of mRNA changes the identity of encoded amino acids and may create translation initiation and termination codons. The existence of RNA editing violates one of the long-accepted tenets of genetic information flow, namely, that the amino acid sequence of a protein can be directly predicted from the corresponding gene sequence. Particular RNA editing systems display a narrow phylogenetic distribution, which argues that such systems are derived within specific eukaryotic lineages, rather than representing traits that ultimately trace to a common ancestor of eukaryotes, or even further back in evolution. The derived nature of RNA editing raises intriguing questions about how and why RNA editing systems arise, and how they become fixed as additional, essential steps in genetic information transfer.