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

Abstract

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...

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

“…However, using oligo(dT)-based RT-PCR approaches, we and others (22)(23)(24)(25) have shown that mRNAs may be polyadenylated at low levels. The use of the cRT-PCR technique allows in theory the analysis of both the 3Ј-and 5Ј-ends of transcripts regardless of the polyadenylation status of the transcripts.…”

Transcription and RNA-processing in fission yeast mitochondria

“…In addition, in animals, plants, and in certain protist mitochondria, polyadenylation regulates mRNA stability (for review, see Gagliardi et al 2004). Apparently, mitochondrial polyadenylation is absent in S. cerevisiae (Gagliardi and Leaver 1999;Gagliardi et al 2001).…”

Mitochondrial mRNA stability and polyadenylation during anoxia-induced quiescence in the brine shrimpArtemia franciscana

“…Rather, in these less complicated systems, polyadenylation of mitochondrial message might provide an organizing center for nuclease(s) to begin digesting the 3′ end. Recent work in potato mitochondria (Gagliardi et al, 2001) provides support for the idea that 3′ polyadenylation promotes 3′-5′ degradation. While differences in the mechanism of degradation may exist between plant and animal mitochondria on one hand, and chloroplasts and eubacteria on the other, the relationship between elevated polyadenylation and increased degradation appears to have been conserved between these groups (Gagliardi et al, 2001).…”

“…Recent work in potato mitochondria (Gagliardi et al, 2001) provides support for the idea that 3′ polyadenylation promotes 3′-5′ degradation. While differences in the mechanism of degradation may exist between plant and animal mitochondria on one hand, and chloroplasts and eubacteria on the other, the relationship between elevated polyadenylation and increased degradation appears to have been conserved between these groups (Gagliardi et al, 2001). Animal mitochondria typically contain very little non-coding DNA and have extremely compacted coding regions, while chloroplasts and plant mitochondria are generally much larger and contain genes not found in animal mtDNA, relatively more noncoding Stability and polyadenylation of mRNA during quiescence DNA, and intergenic spacers and UTRs that could serve as cisprocessing signals (Scheffler, 1999).…”