Polyadenylation Occurs at Multiple Sites in Maize Mitochondrial cox2 mRNA and Is Independent of Editing Status

Abstract

Polyadenylation of nucleus-encoded transcripts has a well-defined role in gene expression. The extent and function of polyadenylation in organelles and prokaryotic systems, however, are less well documented. Recent reports of polyadenylation-mediated RNA destabilization in Escherichia coli and in vascular plant chloroplasts prompted us to look for polyadenylation in plant mitochondria. Here, we report the use of reverse transcription-polymerase chain reaction to map multiple polyadenylate addition sites in maize mitochondrial cox2 transcripts. The lack of sequence conservation surrounding these sites suggests that polyadenylation may occur at many 3 Ј termini created by endoribonucleolytic and/or exoribonucleolytic activities, including those activities involved in 3 Ј end maturation. Endogenous transcripts could be efficiently polyadenylated in vitro by using maize mitochondrial lysates with an activity that added AMP more efficiently than GMP. Polyadenylated substrates were tested for stability in maize mitochondrial S100 extracts, and we found that, compared with nonpolyadenylated RNAs, the polyadenylated substrates were less stable. Taken together with the low abundance of polyadenylated RNAs in maize mitochondria, our results are consistent with a degradation-related process. The fact that polyadenylation does not dramatically destabilize plant mitochondrial transcripts, at least in vitro, is in agreement with results obtained for animal mitochondria but differs from those obtained for chloroplasts and E. coli . Because fully edited, partially edited, and unedited transcripts were found among the cloned polyadenylated cox2 cDNAs, we conclude that RNA editing and polyadenylation are independent processes in maize mitochondria. INTRODUCTIONPresent-day mitochondria have almost certainly evolved from a prokaryotic endosymbiont (reviewed in Gray, 1992). These organelles possess their own genomes and gene expression machinery; however, during evolution, most of the genetic information of the mitochondrial ancestor was transferred to the nuclear genome. Mitochondrial genes in Saccharomyces cerevisiae and metazoans are transcribed by a nucleus-encoded T7-like RNA polymerase and accessory factors (reviewed in Tracy and Stern, 1995), and candidate plant nuclear genes encoding mitochondrial RNA polymerase have been identified (Cermakian et al., 1996; Hedtke et al., 1997;Young et al., 1998; Chang et al., 1999). In plants, promoter strength may play a regulatory role in gene expression (Mulligan et al., 1991), but post-transcriptional regulation also can occur by differential RNA stability (Finnegan and Brown, 1990).The maize mitochondrial genome is typical of those found in vascular plants. It can be genetically mapped as a single circular molecule of 570 kb, with multiple repeated sequences giving rise to a variety of stably inherited subgenomic recombination products (Lonsdale et al., 1984). As in other species, maize mitochondrial primary transcripts are subject to both cis -splicing (e.g., Fox and Leaver, 1981) ...

Mapping of mitochondrial mRNA termini in Arabidopsis thaliana : t-elements contribute to 5′ and 3′ end formation

“…Mostly only single adenosines are found and less frequently cytidines, only in rare instances thymidines (corresponding to uridines in the RNA) or guanosines. The extensions can be grouped into two categories as observed previously (18,22,23,46). They are either oligohomopolymeric adenosine stretches (up to 24 adenosines) or short extensions of adenosines and cytosines.…”

“…Another important ( cis )-factor is the polyadenylation state on an RNA. Short oligo(A) tails at mature 3 ′ ends have been found to destabilize plant mitochondrial RNA both in vivo and in vitro and are thus expected only at a minor fraction of the steady state pool (9,18,22,23). …”

Mitochondrial cytochrome c oxidase and succinate dehydrogenase complexes contain plant specific subunits

“…They are almost always localized on the mitochondrial genome and were each first sequenced about 20 years ago (Fox and Leaver, 1981;Isaac et al, 1985;Hiesel et al, 1987). Careful investigation of these genes and their transcription led to the discovery of introns in plant mitochondrial genes (Fox and Leaver, 1981), editing of transcripts in plant mitochondria (Covello and Gray, 1989;Gualberto et al, 1989;Hiesel et al, 1989) and polyadenylation of mitochondrial transcripts (Lupold et al, 1999). Earlier electrophoretic analyses of chromatographically purified cytochrome c oxidase from sweet potato had revealed five protein bands of about 39 kDa (band I), 33 kDa (II), 26 kDa (III), 20 kDa (IV), and 6 kDa (V) (Maeshima and Asahi, 1978).…”

Post-transcriptional Control of Chloroplast Gene Expression

“…In chloroplast extracts, polyadenylated RNAs are degraded faster than nonadenylated RNAs and are more abundant in vivo under specific conditions that promote RNA degradation. Thus, polyadenylation might promote plastid RNA turnover in vivo by targeting endonucleolytic cleavage products for degradation63–66 as described for bacteria67–70 and plant mitochondria 71,72. The molecular mechanism of RNA degradation in chloroplasts appears very similar to that of bacteria 63,64,73.…”