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

Abstract

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 ...

Human Mitochondrial mRNAs Are Stabilized with Polyadenylation Regulated by Mitochondria-specific Poly(A) Polymerase and Polynucleotide Phosphorylase

“…Therefore, hmtPAP is a key enzyme regulating both translation termination and mRNA metabolism in human mitochondria. In contrast, hPNPase acts degradatively, similar to E. coli or Arabidopsis organellar PNPases (6,37,49), and thus controls the extent of mitochondrial polyadenylation. However, it is also shown that hPNPase has in vitro PAP activity (Fig.…”

RNA Processing Factor 7 and Polynucleotide Phosphorylase Are Necessary for Processing and Stability of nad2 mRNA in Arabidopsis Mitochondria

“…In plant mitochondria, PNPase, a 3′ to 5′ exoribonuclease, is involved in the generation of mature 3′ termini of transcripts such as atp9 mRNA but is also required for the removal of RNAs from the total mitochondrial transcript pool. 21,22 To check whether there is any connection between 5′ processing and the degradation of non-processed RNAs by PNPase, we established a transgenic Arabidopsis line expressing an artifical microRNA (amiRNA) targeting the corresponding mRNA (amiR-PNP-3, Fig. S6A).…”

“…Two enzymes were found to be involved in 3′ to 5′ exonucleolytic trimming of the mature 3′ ends. 21,22 The mitochondrial polynucleotide phosphorylase (PNPase) is capable to degrade large parts of the precursor transcript downstream of the mature 3′ terminus, whereas the fine-tuning of the 3′ termini is done by RNase R1 homolog (RNR1), which removes a few nucleotides left over by PNPase. Analogous to the plastid-type transcript processing, the P-class PPR protein MTSF1 binds to the 3′ terminal part of nad4 transcript and it is highly likely that this interaction impedes progression of the 3′ exonucleolytic activities and thus determines the mature 3′ end of this mRNA.…”

Mitochondrial mRNA Polymorphisms in Different Arabidopsis Accessions

“…For instance, two 3# to 5# exoribonucleases involved in the generation of 3# ends (RNR1 and PNPase) have been characterized by reverse-genetics approaches and, very recently, a similar approach identified a protein required for trans-splicing of nad1 transcripts in Arabidopsis (Arabidopsis thaliana) mitochondria (Perrin et al, 2004a(Perrin et al, , 2004bFalcon de Longevialle et al, 2007). Forward-genetics approaches have also been applied to identify genes for nuclear-encoded restorers of fertility in several cytoplasmic male sterility (CMS) systems.…”

“…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.…”