cis Recognition Elements in Plant Mitochondrion RNA Editing

Abstract

RNA editing in higher plant mitochondria modifies mRNA sequences by means of C-to-U conversions at highly specific sites. To determine the cis elements involved in recognition of an editing site in plant mitochondria, deletion and site-directed mutation constructs containing the cognate cox II mitochondrial gene were introduced into purified mitochondria by electroporation. The RNA editing status was analyzed for precursor and spliced transcripts from the test construct. We found that only a restricted number of nucleotides in the vicinity of the target C residue were necessary for recognition by the editing machinery and that the nearest neighbor 3 residues were crucial for the editing process. We provide evidence that two functionally distinguishable sequences can be defined: the 16-nucleotide 5 region, which can be replaced with the same region from another editing site, and a 6-nucleotide 3 region specific to the editing site. The latter region may play a role in positioning the actual editing residue.RNA editing refers to a process whereby the genetic message is changed at single nucleotides in a very specific manner. This process involves a variety of genetic systems and occurs by different mechanisms (reference 6 and references therein). In trypanosome kinetoplasts, RNA editing proceeds by insertion and deletion of uridine nucleotides in mRNAs (2); the insertion of C residues has been described for Physarum polycephalum mitochondria, and the insertion of some G residues occurs in paramyxovirus (25,36). Another type of RNA editing is base conversion, occurring in mammalian nuclei (31) and plant organelles. C-to-U conversions have been described for higher plant mitochondria (9, 13, 16) and to a lesser extent for chloroplasts (18,21).RNA editing is a posttranscriptional event in plant organelles. It is essential in plant mitochondrion gene expression processes such as the maturation step of organellar transcripts (26,27) or the synthesis of functional proteins, since the nucleotide conversions usually alter the coding properties of the mRNA (1). The editing systems in higher plant organelles, mitochondria, and chloroplasts share many similar features, but promiscuous chloroplast sequences are not edited in mitochondria (39); conversely, a mitochondrial sequence carrying an editing site does not sustain editing when transcribed into chloroplasts (35). These results indicate that editing recognition signals are specific to each organelle. The sequences flanking target C residues lack any apparent conserved consensus elements at the primary or secondary structure level. An essential problem is to define the signals that determine the specific recognition of every editing site.A number of in vivo studies of transgenic chloroplasts have demonstrated that mRNA sequences flanking the editing site are involved in RNA editing (4, 5). RNA editing has been determined to proceed by deamination of the C residue in wheat (3) and pea (38). However, the molecular determinants for editing-site recognition have not yet been ...

In Vitro RNA Editing in Pea Mitochondria Requires NTP or dNTP, Suggesting Involvement of an RNA Helicase

“…7). Such comparisons suggested that upstream (5Ј) sequences are crucial in targeting the editing machinery and that downstream similarities are not sufficient to specify a site.These observations were corroborated by the recent breakthrough development of an in vitro editing system for plastids (8, 9) and an electroporation protocol for mitochondria (10,11). Mutational analysis of templates in these as well as in in vivo experiments in transgenic chloroplasts (12)(13)(14) showed that for several editing sites 20 -30 nucleotides upstream and 2-5 nucleotides downstream are sufficient to guide the editing activity, precisely what had initially been deduced for mitochondria (7).…”

A site-specific factor interacts directly with its cognate RNA editing site in chloroplast transcripts

“…Consistent with these observations, conversion to G abolished both psbE and petB mRNA editing in vitro. Similarly, the conversion of 5Ј neighboring residues to G in chloroplast ndhB (site V) mRNA and mitochondrial cox II (C259) mRNA were reported to inhibit editing in vivo (13,24). A guanosine at the 5Ј neighboring position is inhibitory for most editing sites studied; therefore, the strong bias against G may be due to mechanical constraints.…”

RNA editing sites in plant mitochondria can share cis‐elements

“…The development of reliable in vitro RNA editing activities for chloroplasts [7][8][9] and mitochondria [10,11] as well as in organello editing [12][13][14][15] in the past few years, has accelerated progress towards elucidating the cis-requirements. For plant mitochondria, in vitro RNA editing in pea lysates and in organello editing in wheat show that for some editing events only about 15-30 nucleotides are necessary upstream and very few or none downstream.…”