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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 two-step 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.

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.