Transcript Lifetime Is Balanced between Stabilizing Stem-Loop Structures and Degradation-Promoting Polyadenylation in Plant Mitochondria

100

In plant mitochondria, transcription proceeds well beyond the region that will become mature 3 extremities of mRNAs, and the mechanisms of 3 maturation are largely unknown. Here, we show the involvement of two exoribonucleases, AtmtPNPase and AtmtRNaseII, in the 3 processing of atp9 mRNAs in Arabidopsis thaliana mitochondria. Down-regulation of AtmtPNPase results in the accumulation of pretranscripts of several times the size of mature atp9 mRNAs, indicating that 3 processing of these transcripts is performed mainly exonucleolytically by AtmtPNPase. This enzyme is however not sufficient to completely process atp9 mRNAs, because with down-regulation of another mitochondrial exoribonuclease, AtmtRNaseII, about half of atp9 transcripts exhibit short 3 nucleotide extensions compared with mature mRNAs. These short extensions can be efficiently removed by AtmtRNaseII in vitro. Taken together, these results show that 3 processing of atp9 mRNAs in Arabidopsis mitochondria is, at least, a twostep phenomenon. First, AtmtPNPase is involved in removing 3 extensions that may reach several kilobases. Second, AtmtRNaseII degrades short nucleotidic extensions to generate the mature 3 -ends.Functional RNAs are generated through a series of complex post-transcriptional processes. The nature of these processes can vary depending on the organism or cellular compartment considered and may include maturation of 5Ј and 3Ј termini, splicing of introns, capping, editing, base modifications, or addition of nongenomically-encoded nucleotides such as polyadenylation. Processing of mRNAs is well documented for bacterial and eukaryotic nuclear mRNAs (1, 2). Our knowledge of maturation of plastid transcripts is also extensive for both Chlamydomonas and higher plants (3). However, mRNA maturation processes in plant mitochondria are far less well characterized. Two lines of evidence suggest that processing of 3Ј extremities occurs for any RNA in plant mitochondria. First, run-on experiments have demonstrated that transcription proceeds beyond the region that corresponds to mature 3Ј-ends of mRNAs and rRNAs (4). Second, it has been shown using an in vitro transcription system that inverted repeats that terminate some mitochondrial mRNAs are not recognized as transcription termination signals (5). These inverted repeats seem to be essential as processing signals in vitro (5) and in vivo (6, 7). However, the ribonucleases involved in 3Ј mRNA maturation processes remain to be identified in plant mitochondria.Escherichia coli represents one of the model organisms for which the function, mechanism, and regulation of ribonucleases (RNases) are intensively studied (for example, see Refs. 1, 8, and 9). Among the eight characterized exoribonucleases in E. coli, polynucleotide phosphorylase (PNPase) 1 and RNaseII are the main exoribonucleases involved in degrading mRNAs in a 3Ј to 5Ј direction but participate also in processing events. PNPase-like proteins are also present in human mitochondria (10) and in chloroplasts. In the latter, it is involved both ...

Section: Resultsmentioning

confidence: 99%

Two Exoribonucleases Act Sequentially to Process Mature 3′-Ends of atp9 mRNAs in Arabidopsis Mitochondria

Perrin

Meyer

Zaepfel

et al. 2004

100

“…The pea mitochondrial atp9 gene was chosen as standard substrate because this gene is abundantly transcribed and has been investigated in detail with respect to its promoters and transcript stability (22,23). It is furthermore efficiently edited at most of its editing sites, 2 including the first site in the reading frame, which was selected as the editing target.…”

Section: The In Vitro Editingmentioning

confidence: 99%

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

Takenaka

Brennicke

2003

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...

“…Polyadenylation is required for stabilizing virtually all nuclear-encoded mRNAs in all eukaryotes. In contrast, polyadenylation targets mRNAs for degradation in eubacteria (1,2), chloroplasts (3)(4)(5)(6), plant mitochondria (7)(8)(9), and trypanosome mitochondria (10). In eubacteria and chloroplasts, degradation of mRNAs is initiated by endonucleolytic cleavages.…”

mentioning

confidence: 99%

“…Maize cox2 mRNAs may also be polyadenylated, although the influence of polyadenylation on the degradation rate in vitro is less pronounced (8). Recently, polyadenylation of pea atp9 and Oenothera atp1 transcripts has also been characterized (9). Both transcripts terminate with a stable double stem-loop structure, which compensates to some extent for the destabilization effect of polyadenylation in vitro (9).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Plant Mitochondrial Polyadenylated mRNAs Are Degraded by a 3′- to 5′-Exoribonuclease Activity, Which Proceeds Unimpeded by Stable Secondary Structures

Gagliardi¹,

Perrin²,

Maréchal-Drouard³

et al. 2001

Recently, we and others have reported that mRNAs may be polyadenylated in plant mitochondria, and that polyadenylation accelerates the degradation rate of mRNAs. To further characterize the molecular mechanisms involved in plant mitochondrial mRNA degradation, we have analyzed the polyadenylation and degradation processes of potato atp9 mRNAs. The overall majority of polyadenylation sites of potato atp9 mRNAs is located at or in the vicinity of their mature 3-extremities. We show that a 3-to 5-exoribonuclease activity is responsible for the preferential degradation of polyadenylated mRNAs as compared with non-polyadenylated mRNAs, and that 20 -30 adenosine residues constitute the optimal poly(A) tail size for inducing degradation of RNA substrates in vitro. The addition of as few as seven non-adenosine nucleotides 3 to the poly(A) tail is sufficient to almost completely inhibit the in vitro degradation of the RNA substrate. Interestingly, the exoribonuclease activity proceeds unimpeded by stable secondary structures present in RNA substrates. From these results, we propose that in plant mitochondria, poly(A) tails added at the 3 ends of mRNAs promote an efficient 3-to 5-degradation process.The control of mRNA stability constitutes an important aspect of the regulation of gene expression in all organisms. Polyadenylation of mRNAs is involved in the control of mRNA stability, however, with two significantly different roles depending on the organism or subcellular compartment concerned. Polyadenylation is required for stabilizing virtually all nuclear-encoded mRNAs in all eukaryotes. In contrast, polyadenylation targets mRNAs for degradation in eubacteria (1, 2), chloroplasts (3-6), plant mitochondria (7-9), and trypanosome mitochondria (10). In eubacteria and chloroplasts, degradation of mRNAs is initiated by endonucleolytic cleavages. The resulting fragments are then degraded by 3Ј-to 5Ј-exonuclease activities (1, 5). In both systems, polyadenylation targets endonucleolytic cleavage products for rapid degradation. In chloroplasts, structured mature 3Ј ends are poor substrates for polyadenylation as compared with the internal RNA fragments generated by endonuclease(s) (5, 6).In plant mitochondria, molecular mechanisms involved in mRNA stability or degradation are still poorly understood (11). For instance, no proteins involved in these processes have been formally identified, although several candidate genes have now been identified since the completion of the nuclear genome sequence of Arabidopsis thaliana. Stable secondary structures can be predicted at the 3Ј-extremities of some but not all plant mitochondrial mRNAs (12). When present, these structures appear to be involved in stabilizing the transcripts (13-15) and in the correct processing of 3Ј-extremities rather than being signals for transcription termination (15). Mature 3Ј termini of plant mitochondria mRNAs are generated by 3Ј-processing of longer pre-mRNA molecules (15), and stable mature mRNAs are not constitutively polyadenylated at their 3Ј-extremit...