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

Abstract

To analyze the biochemical parameters of RNA editing in plant mitochondria and to eventually characterize the enzymes involved we developed a novel in vitro system. The high sensitivity of the mismatch-specific thymine glycosylase is exploited to facilitate reliable quantitative evaluation of the in vitro RNA editing products. A pea mitochondrial lysate correctly processes a C to U editing site in the cognate atp9 template. Reaction conditions were determined for a number of parameters, which allow first conclusions on the proteins involved. The apparent tolerance against specific Zn 2؉ chelators argues against the involvement of a cytidine deaminase enzyme, the theoretically most straightforward catalysator of the deamination reaction. Participation of a transaminase was investigated by testing potential amino group receptors, but none of these increased the RNA editing reaction. Most notable is the requirement of the RNA editing activity for NTPs. Any NTP or dNTP can substitute for ATP to the optimal concentration of 15 mM. This observation suggests the participation of an RNA helicase in the predicted RNA editing protein complex of plant mitochondria.RNA editing has been observed in various forms in diverse organisms including trypanosomes, mammals, slime molds, and land plants (1). In land plants, RNA editing has so far been reported in mitochondria and plastids. In flowering plants the majority of this organellar editing involves C to U conversions, with U to C changes occurring much more infrequently and only in mitochondria (2, 3, 4). In non-flowering plants this latter direction of RNA editing is much more common in mitochondria as well as in chloroplasts, the U to C reactions almost reaching the frequency of the C to U editing in the hornworts (5). Since its discovery more than a decade ago, progress into understanding how this process works has been hampered by the lack of manageable and reliable in vitro systems. The first in vitro system for plant mitochondrial RNA editing was successfully developed from wheat embryos (6), but has not been exploited further.Conclusions about the determinants of specificity during RNA editing in mitochondria and in plastids have initially been drawn from comparisons of transcribed sequence duplications (e.g. 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).The biochemistry of this RNA editing activity is still largely unclear, and more detailed u...

RNA editing sites in plant mitochondria can share cis‐elements

“…The RNA editing product percentages lie well within the range of the linear sensitivity of the TDG assay as determined experimentally and the relative amounts of cut fragments (3-7%) thus reflect the amount of the in vitro editing activity within this window (about 1-25% [10]). …”

“…DNA clones (patp9) were constructed in an adapted pBluescript SK+ vector to allow run-off transcription of the editing template RNA as described [10]. Deletion clones were shortened by removing original mitochondrial sequences as indicated in the respective experiments.…”

“…The in vitro RNA editing reactions were performed as described [10]. After incubation, template sequences were amplified by RT-PCR, the upstream primer labelled with the Cy5 fluorophor.…”

“…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.…”

Mitochondrial mRNA Polymorphisms in Different Arabidopsis Accessions

“…Detailed investigations of these posttranscriptional processes are still at the beginning and up to now have been predominantly descriptive. Although functional studies using in vitro or in organello systems have allowed some insight into the mechanisms of RNA editing, splicing, or 3# end formation Araya, 2001, 2002;Staudinger and Kempken, 2003;Takenaka and Brennicke, 2003;Perrin et al, 2004aPerrin et al, , 2004b, still very little is known about the trans-factors active in these processes. One of the least explored maturation steps is the generation of mature 5# ends of mitochondrial mRNAs.…”