Distinct roles for sequences upstream of and downstream from Physarum editing sites

RNAs in the mitochondria of Physarum polycephalum contain nonencoded nucleotides that are added during RNA synthesis. Essentially all steady-state RNAs are accurately and fully edited, yet the signals guiding these precise nucleotide insertions are presently unknown. To localize the regions of the template that are required for editing, we constructed a series of chimeric templates that substitute varying amounts of DNA either upstream of or downstream from C insertion sites. Remarkably, all sequences necessary for C addition are contained within ∼9 base pairs on either side of the insertion site. In addition, our data strongly suggest that sequences within this critical region affect different steps in the editing reaction. Template alterations upstream of an editing site influence nucleotide selection and/or insertion, while downstream changes affect editing site recognition and templated extension from the added, unpaired nucleotide. The data presented here provide the first evidence that individual regions of the DNA template play discrete mechanistic roles and represent a crucial initial step toward defining the source of the editing specificity in Physarum mitochondria. In addition, these findings have mechanistic implications regarding the potential involvement of the mitochondrial RNA polymerase in the editing reaction.     

Accurate and efficient insertional RNA editing in isolated Physarum mitochondria.

RNA editing is a process whereby nucleotide insertion, deletion, or base substitution results in the production of an RNA whose sequence differs from that of its template. The mitochondrial RNAs of Physarum polycephalum are processed specifically at multiple sites by both mono- and dinucleotide insertions, as well as apparent cytidine (C) to uridine (U) changes. The precise mechanism and timing of these processing events are currently unknown. We describe here the development of an isolated mitochondrial system in which exogenously supplied nucleotides can be incorporated into RNAs under defined conditions. The results of S1 nuclease protection, nearest neighbor and RNase T1 fingerprint analyses indicate that the vast majority of these newly synthesized mitochondrial RNAs have been accurately and efficiently processed by both mono- and dinucleotide insertions. This work provides a direct demonstration of faithful nucleotide insertion in a mitochondrial editing system. In contrast, the newly synthesized RNAs are not processed by C to U changes in the isolated mitochondria, suggesting that the base changes observed in Physarum are unlikely to occur via a deletion/insertion mechanism.     

Partially edited RNAs are intermediates of RNA editing in plant mitochondria

RNA editing in flowering plant mitochondria addresses several hundred specific C nucleotides in individual sequence contexts in mRNAs and tRNAs. Many of the in vivo steady state RNAs are edited at some sites but not at others. It is still unclear whether such incompletely edited RNAs can either be completed or are aborted. To learn more about the dynamics of the substrate recognition process, we investigated in vitro RNA editing at a locus in the atp4 mRNA where three editing sites are clustered within four nucleotides. A single cis-element of about 20 nucleotides serves in the recognition of at least two sites. Competition with this sequence element suppresses in vitro editing. Surprisingly, unedited and edited competitors are equally effective. Experiments with partially pre-edited substrates indicate that indeed the editing status of a substrate RNA does not affect the binding affinity of the specificity factor(s). RNA molecules in which all editing sites are substituted by either A or G still compete, confirming that editing site recognition can occur independently of the actual editing site. These results show that incompletely edited mRNAs can be substrates for further rounds of RNA editing, resolving a long debated question.     

RNA editing of the col mRNA throughout the life cycle of Physarum polycephalum

Editing of RNA via the insertion, deletion or substitution of genetic information affects gene expression in a variety of systems. Previous characterization of the Physarum polycephalum cytochrome c oxidase subunit I (col) mRNA revealed that both nucleotide insertions and base substitutions occur during the maturation of this mitochondrial message. Both types of editing are known to be developmentally regulated in other systems, including mammals and trypanosomatids. Here we show that the col mRNA present in Physarum mitochondria is edited via specific nucleotide insertions and C to U conversions at every stage of the life cycle. Primer extension sequencing of the RNA indicates that this editing is both accurate and efficient. Using a sensitive RT-PCR assay to monitor the extent of editing at individual sites of C insertion, we estimate that greater than 98% of the steady-state amount of col mRNA is edited throughout the Physarum developmental cycle.     

RNA editing of the col mRNA throughout the life cycle of Physarum polycephalum

Editing of RNA via the insertion, deletion or substitution of genetic information affects gene expression in a variety of systems. Previous characterization of the Physarum polycephalum cytochrome c oxidase subunit I (col) mRNA revealed that both nucleotide insertions and base substitutions occur during the maturation of this mitochondrial message. Both types of editing are known to be developmentally regulated in other systems, including mammals and trypanosomatids. Here we show that the col mRNA present in Physarum mitochondria is edited via specific nucleotide insertions and C to U conversions at every stage of the life cycle. Primer extension sequencing of the RNA indicates that this editing is both accurate and efficient. Using a sensitive RT-PCR assay to monitor the extent of editing at individual sites of C insertion, we estimate that greater than 98% of the steady-state amount of col mRNA is edited throughout the Physarum developmental cycle.     

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.     

Insertional editing in mitochondria of Physarum

RNA produced from a number of genes on the mitochondrial (mt) DNA of Physarum polycephalum have nucleotides inserted at specific sites in their sequence. These insertions are spaced at approximately 25 nucleotide intervals and create open reading frames in mRNA and functional structure in tRNAs and rRNAs. Although most of the insertions at a site are single cytidines; single uridines and certain dinucleotides containing adenosine and guanosine as well as cytidine and uridine are also occasionally inserted at certain sites. This mixed nucleotide insertional RNA editing is unique among currently characterized editing systems.