AtmtPNPase is required for multiple aspects of the 18S rRNA metabolism in Arabidopsis thaliana mitochondria

Abstract

Plant mitochondria contain three rRNA genes, rrn26, rrn18 and rrn5, the latter two being co-transcribed. We have recently identified a polynucleotide phosphorylase-like protein (AtmtPNPase) in Arabidopsis mitochondria. Plants downregulated for AtmtPNPase expression (PNP-plants) accumulate 18S rRNA species polyadenylated at internal sites, indicating that AtmtPNPase is involved in 18S rRNA degradation. In addition, AtmtPNPase is required to degrade the leader sequence of 18S rRNA, a maturation by-product excised by an endonucleolytic cut 5' to the 18S rRNA. PNP-plants also accumulate 18S rRNA precursors correctly processed at their 5' end but containing the intergenic sequence (ITS) between the 18S and 5S rRNA. Interestingly, these precursors may be polyadenylated. Taken together, these results suggest that AtmtPNPase initiates the degradation of the ITS from 18S precursors following polyadenylation. To test this, we overexpressed in planta a second mitochondrial exoribonuclease, AtmtRNaseII, that degrades efficiently unstructured RNA including poly(A) tails. This resulted also in the detection of 18S rRNA precursors showing that AtmtRNaseII is not able to degrade the ITS but can impede the action of AtmtPNPase in initiating the degradation of the ITS. These results show that AtmtPNPase is essential for several aspects of 18S rRNA metabolism in Arabidopsis mitochondria.

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).…”

“…S7). Since previously established PNPase co-suppression lines were found to be impaired in atp9 mRNA 3′ processing, 21,22 we analyzed this transcript in the amiR-PNP-3 plants. Both CR-RT-PCR and RT-PCR indicated excessive amounts of 3′ extended atp9 transcripts in PNPase knockdown plants (Fig.…”

“…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

“…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.…”

“…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.…”