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Viral RNAs can be uridylated in eucaryotic hosts. However, our knowledge of uridylation patterns and roles remains rudimentary for phytoviruses. Here, we report global 3’ terminal RNA uridylation profiles for representatives of the main families of positive single-stranded RNA phytoviruses. We detected uridylation in all 47 viral RNAs investigated here, revealing its prevalence. Yet, uridylation levels of viral RNAs varied from 0.2% to 90%. Unexpectedly, most poly(A) tails of grapevine fanleaf virus (GFLV) RNAs, including encapsidated tails, were strictly mono-uridylated, which corresponded to an unidentified type of viral genomic RNA extremity. This mono-uridylation appears beneficial for GFLV because it becomes dominant when plants are infected with non-uridylated GFLV transcripts. We found that GFLV RNA mono-uridylation is independent of the known TUTases HEN1 SUPPRESSOR 1 (HESO1) and UTP:RNA URIDYLYLTRANSFERASE 1 (URT1) in Arabidopsis (Arabidopsis thaliana). By contrast, both TUTases uridylate other viral RNAs like turnip crinkle virus (TCV) and turnip mosaic virus (TuMV) RNAs. Interestingly, TCV and TuMV degradation intermediates were differentially uridylated by HESO1 and URT1. Although the lack of both TUTases did not prevent viral infection, we detected degradation intermediates of TCV RNA at higher levels in a Arabidopsis heso1 urt1 mutant, suggesting that uridylation participates in clearing viral RNA. Collectively, our work unveils an extreme diversity of uridylation patterns across phytoviruses and constitutes a valuable resource to further decipher pro- and anti-viral roles of uridylation.

In eukaryotes, general mRNA decay requires the decapping complex. The activity of this complex depends on its catalytic subunit, DECAPPING2 (DCP2), and its interaction with decapping enhancers, including its main partner DECAPPING1 (DCP1). Here, we report that in Arabidopsis thaliana, DCP1 also interacts with a NYN domain endoribonuclease, hence named DCP1-ASSOCIATED NYN ENDORIBONUCLEASE 1 (DNE1). Interestingly, we found DNE1 predominantly associated with DCP1, but not with DCP2, and reciprocally, suggesting the existence of two distinct protein complexes. We also showed that the catalytic residues of DNE1 are required to repress the expression of mRNAs in planta upon transient expression. The overexpression of DNE1 in transgenic lines led to growth defects and a similar gene deregulation signature than inactivation of the decapping complex. Finally, the combination of dne1 and dcp2 mutations revealed a functional redundancy between DNE1 and DCP2 in controlling phyllotactic pattern formation. Our work identifies DNE1, a hitherto unknown DCP1 protein partner highly conserved in the plant kingdom and identifies its importance for developmental robustness.

SERRATE/ARS2 is a conserved RNA effector protein involved in transcription, processing and export of different types of RNAs. In Arabidopsis, the best-studied function of SERRATE (SE) is to promote miRNA processing. Here, we report that SE interacts with the Nuclear Exosome Targeting (NEXT) complex, comprising the RNA helicase HEN2, the RNA binding protein RBM7 and one of the two zinc-knuckle proteins ZCCHC8A/ZCCHC8B. The identification of common targets of SE and HEN2 by RNA-seq supports the idea that SE cooperates with NEXT for RNA surveillance by the nuclear exosome. Among the RNA targets accumulating in absence of SE or NEXT are miRNA precursors. Loss of NEXT components results in the accumulation of pri-miRNAs without affecting levels of miRNAs, indicating that NEXT is, unlike SE, not required for miRNA processing. As compared to se-2, se-2 hen2-2 double mutants showed increased accumulation of pri-miRNAs, but partially restored levels of mature miRNAs and attenuated developmental defects. We propose that the slow degradation of pri-miRNAs caused by loss of HEN2 compensates for the poor miRNA processing efficiency in se-2 mutants, and that SE regulates miRNA biogenesis through its double contribution in promoting miRNA processing but also pri-miRNA degradation through the recruitment of the NEXT complex.

RNA exosome complexes provide the main 3′–5′-exoribonuclease activities in eukaryotic cells and contribute to the maturation and degradation of virtually all types of RNA. RNA exosomes consist of a conserved core complex that associates with exoribonucleases and with multimeric cofactors that recruit the enzyme to its RNA targets. Despite an overall high level of structural and functional conservation, the enzymatic activities and compositions of exosome complexes and their cofactor modules differ among eukaryotes. This review highlights unique features of plant exosome complexes, such as the phosphorolytic activity of the core complex, and discusses the exosome cofactors that operate in plants and are dedicated to the maturation of ribosomal RNA, the elimination of spurious, misprocessed, and superfluous transcripts, or the removal of mRNAs cleaved by the RNA-induced silencing complex and other mRNAs prone to undergo silencing.

In eukaryotes, general mRNA decay requires the decapping complex. The activity of this complex depends on its catalytic subunit, DCP2 and its interaction with decapping enhancers, including its main partner DCP1. Here, we report that in Arabidopsis, DCP1 also interacts with a NYN domain endoribonuclease, hence named DCP1-ASSOCIATED NYN ENDORIBONUCLEASE 1 (DNE1). Interestingly, we find DNE1 predominantly associated with DCP1 but not with DCP2 and reciprocally, suggesting the existence of two distinct protein complexes. We also show that the catalytic residues of DNE1 are required to repress the expression of mRNAs in planta upon transient expression. The overexpression of DNE1 in transgenic lines leads to growth defects and transcriptomic changes related to the one observed upon inactivation of the decapping complex. Finally, the combination of dne1 and dcp2 mutations, revealed a functional redundancy between DNE1 and DCP2 in controlling phyllotactic pattern formation in Arabidopsis. Our work identifies DNE1, a hitherto unknown DCP1 protein partner highly conserved in the plant kingdom and identifies its importance for developmental robustness.One-sentence summaryDNE1, a NYN domain protein interacts with the decapping activator DCP1 and, together with DCP2, specify phyllotactic patterns in Arabidopsis.

The RNA exosome is a key 3’−5’ exoribonuclease with an evolutionarily conserved structure and function. Its cytosolic functions require the co-factors SKI7 and the Ski complex. Here we demonstrate by co-purification experiments that the ARM-repeat protein RESURRECTION1 (RST1) and RST1 INTERACTING PROTEIN (RIPR) connect the cytosolic Arabidopsis RNA exosome to the Ski complex. rst1 and ripr mutants accumulate RNA quality control siRNAs (rqc-siRNAs) produced by the post-transcriptional gene silencing (PTGS) machinery when mRNA degradation is compromised. The small RNA populations observed in rst1 and ripr mutants are also detected in mutants lacking the RRP45B/CER7 core exosome subunit. Thus, molecular and genetic evidence supports a physical and functional link between RST1, RIPR and the RNA exosome. Our data reveal the existence of additional cytosolic exosome co-factors besides the known Ski subunits. RST1 is not restricted to plants, as homologues with a similar domain architecture but unknown function exist in animals, including humans., Cytosolic RNA degradation by the RNA exosome requires the Ski complex. Here the authors show that the proteins RST1 and RIPR assist the RNA exosome and the Ski complex in RNA degradation, thereby preventing the production of secondary siRNAs from endogenous mRNAs.

Uridylation is a widespread modification destabilizing eukaryotic mRNAs. Yet, molecular mechanisms underlying TUTase-mediated mRNA degradation remain mostly unresolved. Here, we report that the Arabidopsis TUTase URT1 participates in a molecular network connecting several translational repressors/decapping activators. URT1 directly interacts with DECAPPING 5 (DCP5), the Arabidopsis ortholog of human LSM14 and yeast Scd6, and this interaction connects URT1 to additional decay factors like DDX6/Dhh1-like RNA helicases. Nanopore direct RNA sequencing reveals a global role of URT1 in shaping poly(A) tail length, notably by preventing the accumulation of excessively deadenylated mRNAs. Based on in vitro and in planta data, we propose a model that explains how URT1 could reduce the accumulation of oligo(A)-tailed mRNAs both by favoring their degradation and because 3’ terminal uridines intrinsically hinder deadenylation. Importantly, preventing the accumulation of excessively deadenylated mRNAs avoids the biogenesis of illegitimate siRNAs that silence endogenous mRNAs and perturb Arabidopsis growth and development., TUTase mediated uridylation of mRNA promotes degradation. Here, Scheer, de Almeida et al. show that Arabidopsis TUTase URT1 interacts directly with the translation inhibitor and decay factor DECAPPING5 and suppresses siRNA biogenesis by preventing accumulation of deadenylated mRNAs

International audience; The main 3'-5' exoribonucleolytic activity of eukaryotic cells is provided by the RNA exosome. The exosome is constituted by a core complex of nine subunits (Exo9), which coordinates the recruitment and the activities of distinct types of cofactors. The RNA exosome cofactors confer distributive and processive 3'-5' exoribonucleolytic, endoribonucleolytic, and RNA helicase activities. In addition, several RNA binding proteins and terminal nucleotidyltransferases also participate in the recognition of exosome RNA substrates.To fully understand the biological roles of the exosome, the respective functions of its cofactors must be deciphered. This entails the high-resolution analysis of 3' extremities of degradation or processing intermediates in different mutant backgrounds or growth conditions. Here, we describe a detailed 3' RACE-seq procedure for targeted mapping of exosome substrate 3' ends. This procedure combines a 3' RACE protocol with Illumina sequencing to enable the high-resolution mapping of 3' extremities and the identification of untemplated nucleotides for selected RNA targets.

Groundbreaking discoveries have uncovered the widespread post-transcriptional modifications of all classes of RNA. These studies have led to the emerging notion of an 'epitranscriptome' as a new layer of gene regulation. Diverse modifications control RNA fate, including the 3' addition of untemplated nucleotides or 3' tailing. The most exciting recent discoveries in 3' tailing are related to uridylation. Uridylation targets various noncoding RNAs, from small RNAs and their precursors to rRNAs, and U tails mostly regulate processing or degradation. Interestingly, uridylation is also a pervasive modification of mRNAs. In this review, we discuss how the addition of few uridines to the 3' end of mRNAs influences mRNA decay. We also consider recent findings that reveal other consequences of uridylation on mRNA fate.

RNA uridylation consists of the untemplated addition of uridines at the 3′ extremity of an RNA molecule. RNA uridylation is catalysed by terminal uridylyltransferases (TUTases), which form a subgroup of the terminal nucleotidyltransferase family, to which poly(A) polymerases also belong. The key role of RNA uridylation is to regulate RNA degradation in a variety of eukaryotes, including fission yeast, plants and animals. In plants, RNA uridylation has been mostly studied in two model species, the green algae Chlamydomonas reinhardtii and the flowering plant Arabidopsis thaliana . Plant TUTases target a variety of RNA substrates, differing in size and function. These RNA substrates include microRNAs (miRNAs), small interfering silencing RNAs (siRNAs), ribosomal RNAs (rRNAs), messenger RNAs (mRNAs) and mRNA fragments generated during post-transcriptional gene silencing. Viral RNAs can also get uridylated during plant infection. We describe here the evolutionary history of plant TUTases and we summarize the diverse molecular functions of uridylation during RNA degradation processes in plants. We also outline key points of future research. This article is part of the theme issue ‘5′ and 3′ modifications controlling RNA degradation’.

International audience; The RNA helicase UP-FRAMESHIFT (UPF1) is a key factor of nonsense-mediated decay (NMD), a mRNA decay pathway involved in RNA quality control and in the fine-tuning of gene expression. UPF1 recruits UPF2 and UPF3 to constitute the NMD core complex, which is conserved across eukaryotes. No other components of UPF1-containing ribonucleoproteins (RNPs) are known in plants, despite its key role in regulating gene expression. Here, we report the identification of a large set of proteins that co-purify with the Arabidopsis UPF1, either in an RNA-dependent or RNA-independent manner. We found that like UPF1, several of its co-purifying proteins have a dual localization in the cytosol and in P-bodies, which are dynamic structures formed by the condensation of translationally repressed mRNPs. Interestingly, more than half of the proteins of the UPF1 interactome also co-purify with DCP5, a conserved translation repressor also involved in P-body formation. We identified a terminal nucleotidyltransferase, ribonucleases and several RNA helicases among the most significantly enriched proteins co-purifying with both UPF1 and DCP5. Among these, RNA helicases are the homologs of DDX6/Dhh1, known as translation repressors in humans and yeast, respectively. Overall, this study reports a large set of proteins associated with the Arabidopsis UPF1 and DCP5, two components of P-bodies, and reveals an extensive interaction network between RNA degradation and translation repression factors. Using this resource, we identified five hitherto unknown components of P-bodies in plants, pointing out the value of this dataset for the identification of proteins potentially involved in translation repression and/or RNA degradation.

The biosynthesis of ribosomal RNA and its incorporation into functional ribosomes is an essential and intricate process that includes production of mature ribosomal RNA from large precursors. Here, we analyse the contribution of the plant exosome and its co-factors to processing and degradation of 18S pre-RNAs in Arabidopsis thaliana. Our data show that, unlike in yeast and humans, an RRP6 homologue, the nucleolar exoribonuclease RRP6L2, and the exosome complex, together with RRP44, function in two distinct steps of pre-18S rRNA processing or degradation in Arabidopsis. In addition, we identify TRL (TRF4/5-like) as the terminal nucleotidyltransferase that is mainly responsible for oligoadenylation of rRNA precursors in Arabidopsis. We show that TRL is required for efficient elimination of the excised 5' external transcribed spacer and of 18S maturation intermediates that escaped 5' processing. Our data also suggest involvement of additional nucleotidyltransferases, including terminal uridylyltransferase(s), in modifying rRNA processing intermediates in plants.

RNA uridylation is a potent and widespread posttranscriptional regulator of gene expression. RNA uridylation has been detected in a range of eukaryotes including trypanosomes, animals, plants, and fungi, but with the noticeable exception of budding yeast. Virtually all classes of eukaryotic RNAs can be uridylated and uridylation can also tag viral RNAs. The untemplated addition of a few uridines at the 3' end of a transcript can have a decisive impact on RNA's fate. In rare instances, uridylation is an intrinsic step in the maturation of noncoding RNAs like for the U6 spliceosomal RNA or mitochondrial guide RNAs in trypanosomes. Uridylation can also switch specific miRNA precursors from a degradative to a processing mode. This switch depends on the number of uridines added which is regulated by the cellular context. Yet, the typical consequence of uridylation on mature noncoding RNAs or their precursors is to accelerate decay. Importantly, mRNAs are also tagged by uridylation. In fact, the advent of novel high throughput sequencing protocols has recently revealed the pervasiveness of mRNA uridylation, from plants to humans. As for noncoding RNAs, the main function to date for mRNA uridylation is to promote degradation. Yet, additional roles begin to be ascribed to U-tailing such as the control of mRNA deadenylation, translation control and possibly storage. All these new findings illustrate that we are just beginning to appreciate the diversity of roles played by RNA uridylation and its full temporal and spatial implication in regulating gene expression. WIREs RNA 2018, 9:e1440. doi: 10.1002/wrna.1440 This article is categorized under: RNA Processing > 3' End Processing RNA Processing > RNA Editing and Modification RNA Turnover and Surveillance > Turnover/Surveillance Mechanisms.

The exosome is a large protein complex mediating 3'-5' RNA degradation in both nucleus and cytosol of all eukaryotic cells. It consists of nine conserved subunits forming the core complex, which associates with ribonucleolytic enzymes and other cofactors such as RNA-binding proteins or RNA helicases. Both the composition of the core exosome and its general role as a major player in RNA maturation, RNA surveillance, and RNA turnover are largely conserved between plants, human, and fungi. However, plant exosomes have some peculiar and interesting features including a catalytically active core subunit, or a certain extent of functional specialization among both core subunits and putative exosome cofactors.

Correct gene expression requires tight RNA quality control both at transcriptional and post-transcriptional levels. Using a splicing-defective allele of PASTICCINO2 (PAS2), a gene essential for plant development, we isolated suppressor mutations modifying pas2-1 mRNA profiles and restoring wild-type growth. Three suppressor of pas2 (sop) mutations modified the degradation of mis-spliced pas2-1 mRNA species, allowing the synthesis of a functional protein. Cloning of the suppressor mutations identified the core subunit of the exosome SOP2/RRP4, the exosome nucleoplasmic cofactor SOP3/HEN2 and a novel zinc-finger protein SOP1 that colocalizes with HEN2 in nucleoplasmic foci. The three SOP proteins counteract post-transcriptional (trans)gene silencing (PTGS), which suggests that they all act in RNA quality control. In addition, sop1 mutants accumulate some, but not all of the misprocessed mRNAs and other types of RNAs that are observed in exosome mutants. Taken together, our data show that SOP1 is a new component of nuclear RNA surveillance that is required for the degradation of a specific subset of nuclear exosome targets., Author Summary Cells use various RNA quality control mechanisms to monitore the correct expression of their genome. Indeed, gene transcription can often generate faulty transcripts that are rapidly degraded to avoid possible deleterious effects to the cell. RNA degradation by the exosome is the main pathway for the removal of unwanted RNA in all kingdoms. Recognition of aberrant RNA involves a number of RNA binding proteins and other factors that target them for degradation by the exosome. Here, we used a genetic approach to identify proteins involved in the degradation of a mis-spliced RNA by the nuclear exosome in plants. Our screen identified two known components of nuclear RNA degradation pathway, namely the exosome core subunit RRP4 and the exosome-associated RNA helicase HEN2 that is required for the elimination of non-ribosomal RNAs by the nuclear exosome. Furthermore, we identified SOP1 as a novel putative exosome cofactor that is required for the degradation of some, but not all, of the substrates of HEN2.

Summary Uridylation emerges as a key modification promoting mRNA degradation in eukaryotes. In addition, uridylation by URT1 prevents the accumulation of excessively deadenylated mRNAs in Arabidopsis . Here, we show that the extent of mRNA deadenylation is controlled by URT1. By using TAIL-seq analysis, we demonstrate the prevalence of mRNA uridylation and the existence, at lower frequencies, of mRNA cytidylation and guanylation in Arabidopsis . Both URT1-dependent and URT1-independent types of uridylation co-exist but only URT1-mediated uridylation prevents the accumulation of excessively deadenylated mRNAs. Importantly, uridylation repairs deadenylated extremities to restore the size distribution observed for non-uridylated oligo(A) tails. In vivo and in vitro data indicate that Poly(A) Binding Protein (PABP) binds to uridylated oligo(A) tails and determines the length of U-extensions added by URT1. Taken together, our results uncover a role for uridylation and PABP in repairing mRNA deadenylated ends and reveal that uridylation plays diverse roles in eukaryotic mRNA metabolism.

The exosome is a conserved protein complex that is responsible for essential 3'→5' RNA degradation in both the nucleus and the cytosol. It is composed of a nine-subunit core complex to which co-factors confer both RNA substrate recognition and ribonucleolytic activities. Very few exosome co-factors have been identified in plants. Here, we have characterized a putative RNA helicase, AtMTR4, that is involved in the degradation of several nucleolar exosome substrates in Arabidopsis thaliana. We show that AtMTR4, rather than its closely related protein HEN2, is required for proper rRNA biogenesis in Arabidopsis. AtMTR4 is mostly localized in the nucleolus, a subcellular compartmentalization that is shared with another exosome co-factor, RRP6L2. AtMTR4 and RRP6L2 cooperate in several steps of rRNA maturation and surveillance, such as processing the 5.8S rRNA and removal of rRNA maturation by-products. Interestingly, degradation of the Arabidopsis 5' external transcribed spacer (5' ETS) requires cooperation of both the 5'→3' and 3'→5' exoribonucleolytic pathways. Accumulating AtMTR4 targets give rise to illegitimate small RNAs; however, these do not affect rRNA metabolism or contribute to the phenotype of mtr4 mutants. Plants lacking AtMTR4 are viable but show several developmental defects, including aberrant vein patterning and pointed first leaves. The mtr4 phenotype resembles that of several ribosomal protein and nucleolin mutants, and may be explained by delayed ribosome biogenesis, as we observed a reduced rate of rRNA accumulation in mtr4 mutants. Taken together, these data link AtMTR4 with rRNA biogenesis and development in Arabidopsis.

RNA degradation is a key process in the regulation of gene expression. In all organisms, RNA degradation participates in controlling coding and non-coding RNA levels in response to developmental and environmental cues. RNA degradation is also crucial for the elimination of defective RNAs. Those defective RNAs are mostly produced by ‘mistakes’ made by the RNA processing machinery during the maturation of functional transcripts from their precursors. The constant control of RNA quality prevents potential deleterious effects caused by the accumulation of aberrant non-coding transcripts or by the translation of defective messenger RNAs (mRNAs). Prokaryotic and eukaryotic organisms are also under the constant threat of attacks from pathogens, mostly viruses, and one common line of defence involves the ribonucleolytic digestion of the invader's RNA. Finally, mutations in components involved in RNA degradation are associated with numerous diseases in humans, and this together with the multiplicity of its roles illustrates the biological importance of RNA degradation. RNA degradation is mostly viewed as a default pathway: any functional RNA (including a successful pathogenic RNA) must be protected from the scavenging RNA degradation machinery. Yet, this protection must be temporary, and it will be overcome at one point because the ultimate fate of any cellular RNA is to be eliminated. This special issue focuses on modifications deposited at the 5′ or the 3′ extremities of RNA, and how these modifications control RNA stability or degradation. This article is part of the theme issue ‘5′ and 3′ modifications controlling RNA degradation’.

Small regulatory RNAs are fundamental in eukaryotic and prokaryotic gene regulation. In plants, an important element of post-transcriptional control is effected by 20–24 nt microRNAs (miRNAs) and short interfering RNAs (siRNAs) bound to the ARGONAUTE1 (AGO1) protein in an RNA induced silencing complex (RISC). AGO1 may cleave target mRNAs with small RNA complementarity, but the fate of the resulting cleavage fragments remains incompletely understood. Here, we show that SKI2, SKI3 and SKI8, subunits of a cytoplasmic cofactor of the RNA exosome, are required for degradation of RISC 5′, but not 3′-cleavage fragments in Arabidopsis. In the absence of SKI2 activity, many miRNA targets produce siRNAs via the RNA-dependent RNA polymerase 6 (RDR6) pathway. These siRNAs are low-abundant, and map close to the cleavage site. In most cases, siRNAs were produced 5′ to the cleavage site, but several examples of 3′-spreading were also identified. These observations suggest that siRNAs do not simply derive from RDR6 action on stable 5′-cleavage fragments and hence that SKI2 has a direct role in limiting secondary siRNA production in addition to its function in mediating degradation of 5′-cleavage fragments. ISSN:1362-4962 ISSN:0301-5610

Mitochondria arose from the endosymbiosis of a bacterium related to contemporary members of Alphaproteobacteria (2). Despite a probable monophylogenetic origin and the conservation of most of the biological roles of mitochondria, mitochondrial (mt) genome organization and expression are extraordinarily diverse among eucaryotes. As exemplified in animals and higher plants, mt genome size can vary from 16 kbp to several hundreds of kilobase pairs, respectively. This large increase in plant mt genome size is not correlated with increasing coding capacity but is rather explained by the presence of large intergenic regions, which are virtually absent from animal mt genomes. Plant mt genomes also contain insertions of nuclear and plastid sequences as well as foreign DNA from viral and unknown origins. As a consequence of the diversity in genome organization, mechanisms controlling mt gene expression also differ markedly between organisms. Transcription of the human mt genome is a relatively straightforward process, as both strands are transcribed from single promoters lying in the noncoding regulatory region. By contrast, numerous promoters are scattered along both strands of plant mt genomes. Moreover, even multiple promoters for a single gene are a common feature in plant mitochondria (11, 14). The recent analysis of these promoter sequences in Arabidopsis thaliana also revealed a somehow relaxed promoter specificity, as multiple sequences are able to initiate transcription (11). In addition, there is so far no evidence for a transcription termination mechanism in plant mitochondria. For instance, large regions downstream of rrn genes are expressed, although they do not give rise to stable transcripts (4). The emerging picture is that transcription in plant mitochondria is a relaxed process which, in general, exhibits little control or modulation. Rather, posttranscriptional events such as processing and control of RNA stability account for proper control of gene expression in plant mitochondria (4, 7, 13). Exoribonucleases are major players in RNA 3′ processing and degradation processes. We have recently characterized an A. thaliana nuclear gene (At5g14580) that encodes a mitochondrial exoribonuclease belonging to the polynucleotide phosphorylase (PNPase) family (17). The mt PNPase is essential for viability in Arabidopsis, in contrast to the chloroplast PNPase-like protein encoded by the At3g03710 gene. Down-regulation of the mt PNPase results in the accumulation of unprocessed RNAs, such as atp9 and orfB mRNA and 18S rRNA precursors (16, 17). Moreover, RNA species that are quickly turned over in wild-type (WT) plants accumulate in the absence of PNPase. Such RNAs include, for instance, 18S rRNA degradation intermediates and the leader of the 18S rRNA, which is removed by an endonuclease from the primary transcript. These initial studies revealed that PNPase is essential for several aspects of mt RNA metabolism, including 3′ processing and degradation processes. Interestingly, all RNA substrates of PNPase that we have investigated so far are polyadenylated. Although mature RNAs are not constitutively polyadenylated in plant mitochondria, poly(A) tails trigger rapid exonucleolytic degradation by PNPase, similar to the situation reported first for Escherichia coli and later for chloroplasts (1, 3). To determine new substrates, and thus novel biological roles, of PNPase, we cloned about 300 polyadenylated mt RNAs from plants down-regulated for PNPase (PNP− plants). These sequences were considered degradation tags, as they allowed the identification of different classes of transcripts requiring PNPase for their degradation as judged by their accumulation in PNP− versus WT plants. Our results indicate that maturation by-products such as rRNA leaders or tRNA intergenic sequences are generally degraded by PNPase. In addition, we show that a major role for PNPase is the degradation of transcripts that are, in some cases, expressed to surprisingly high levels from regions lacking known genes. Some of these RNAs, which include transcripts of chimeric open reading frames (ORFs) created by mt DNA recombination events or antisense (AS) RNA transcribed from the opposite DNA strand of a known gene, could have a deleterious effect on mitochondrial function. These data show that the lack of tight transcriptional control in Arabidopsis mitochondria is counterbalanced by polyadenylation-mediated decay by PNPase.

In higher plant mitochondria, post-transcriptional C to U conversion known as editing mostly affects mRNAs. However, three tRNAs were also shown to be edited. Among them, three editing sites were identified in larch mitochondrial tRNAHis. We have previously shown that only the edited version can undergo maturation in vitro. In this paper, we introduced via direct DNA uptake the edited or unedited version of larch mitochondrial trnH into isolated potato mitochondria and expressed them under the control of potato mitochondrial 18 S rRNA promoter. As expected, the edited form of larch mitochondrial tRNAHis precursor was processed in the isolated organelles. By contrast, no mature tRNAHis was detected when using the unedited version of trnH. However, precursor molecules could be characterized by reverse transcription-PCR. These data demonstrate that the potato mitochondrial editing machinery is not able to recognize these “foreign” editing sites and confirm that these unedited tRNA precursor molecules are not correctly processed in organello. As a consequence, the fate of these RNA precursor molecules is likely to be degradation. Indeed, we detected by PCR two 3′-end truncated precursor RNAs. Interestingly, both RNA species exhibit poly(A) tails, a hallmark of degradation in plant mitochondria. Taken together, these data suggest that, in plant mitochondria, a defective unedited RNA precursor that cannot be processed to give a mature stable tRNA, is degraded through a polyadenylation-dependent pathway.

ORFB is the product of a gene that is conserved in plant mitochondrial genomes, and which, on the basis of sequence motif and structural similarity, is predicted to be the homologue of yeast and mammalian ATP8, part of the F(O) component of the F1F(O)-ATP synthase. We have shown that, in sunflower, orfB transcripts are edited, increasing the similarity of the predicted protein to ATP8 proteins from non-plant species. Blue-native polyacrylamide gel electrophoresis and peptide sequencing confirm that ORFB localizes to the ATP synthase complex. The predicted amino-terminal 19 amino acids of ORFB are identical to those in the chimeric mitochondrial ORF522 protein, which is associated with cytoplasmic male sterility (CMS) in sunflower. Assays comparing respiratory complexes from a male-sterile line expressing ORF522 with those from a male-fertile line show a specific decrease in ATP hydrolysis by the ATP synthase. These observations allow us to propose a mechanism underlying CMS that is associated with the expression of chimeric open reading frames containing part of the orfB gene.

Uridine addition at the 3' end of RNAs (i.e., uridylation) emerges as a critical posttranscriptional modification promoting RNA degradation. Uridylation has been notably linked to the degradation of small RNAs, correlated with the 5' shortening of RISC-cleaved transcripts and the degradation of mRNAs. We describe here a method based on 3' RACE (3' Rapid Amplification of cDNA End) PCR that has been successfully used to investigate nucleotide addition at the 3' end of RISC-cleaved transcripts and full-length mRNAs in plants.

The RNA exosome is the major 3′-5′ RNA degradation machine of eukaryotic cells and participates in processing, surveillance and turnover of both nuclear and cytoplasmic RNA. In both yeast and human, all nuclear functions of the exosome require the RNA helicase MTR4. We show that the Arabidopsis core exosome can associate with two related RNA helicases, AtMTR4 and HEN2. Reciprocal co-immunoprecipitation shows that each of the RNA helicases co-purifies with the exosome core complex and with distinct sets of specific proteins. While AtMTR4 is a predominantly nucleolar protein, HEN2 is located in the nucleoplasm and appears to be excluded from nucleoli. We have previously shown that the major role of AtMTR4 is the degradation of rRNA precursors and rRNA maturation by-products. Here, we demonstrate that HEN2 is involved in the degradation of a large number of polyadenylated nuclear exosome substrates such as snoRNA and miRNA precursors, incompletely spliced mRNAs, and spurious transcripts produced from pseudogenes and intergenic regions. Only a weak accumulation of these exosome substrate targets is observed in mtr4 mutants, suggesting that MTR4 can contribute, but plays rather a minor role for the degradation of non-ribosomal RNAs and cryptic transcripts in Arabidopsis. Consistently, transgene post-transcriptional gene silencing (PTGS) is marginally affected in mtr4 mutants, but increased in hen2 mutants, suggesting that it is mostly the nucleoplasmic exosome that degrades aberrant transgene RNAs to limit their entry in the PTGS pathway. Interestingly, HEN2 is conserved throughout green algae, mosses and land plants but absent from metazoans and other eukaryotic lineages. Our data indicate that, in contrast to human and yeast, plants have two functionally specialized RNA helicases that assist the exosome in the degradation of specific nucleolar and nucleoplasmic RNA populations, respectively., Author Summary Cells rely on a number of RNA degradation pathways to ensure correct and timely processing and turnover of both coding and non-coding RNAs. Another important function of RNA degradation is the rapid elimination of misprocessed RNA species, maturation by-products, and nonfunctional RNAs that are frequently produced by pervasive transcription. The main 3′-5′ RNA degradation machine in eukaryotic cells is the exosome, which is activated by cofactors such as RNA helicases. In yeast and human, processing, turnover and surveillance of all nuclear exosome targets depend on a single RNA helicase, MTR4. We show here that the Arabidopsis exosome complex can associate with two related RNA helicases, MTR4 and HEN2. MTR4 and HEN2 reside in nucleolar and nucleoplasmic compartments, respectively, and target different subsets of nuclear RNA substrates for degradation by the exosome. The presence of both MTR4 and HEN2 homologues in green algae, mosses and land plants suggest that the functional duality of exosome-associated RNA helicases is evolutionarily conserved in the entire green lineage. The emerging picture is that, despite a high degree of sequence conservation, intracellular distribution, activities and functions of exosome cofactors vary considerably among different eukaryotes.

Uridine addition at the 3' end of RNAs (i.e., uridylation) emerges as a critical posttranscriptional modification promoting RNA degradation. Uridylation has been notably linked to the degradation of small RNAs, correlated with the 5' shortening of RISC-cleaved transcripts and the degradation of mRNAs. We describe here a method based on 3' RACE (3' Rapid Amplification of cDNA End) PCR that has been successfully used to investigate nucleotide addition at the 3' end of RISC-cleaved transcripts and full-length mRNAs in plants.

In sunflower, PET1-cytoplasmic male sterility is correlated with the presence of a novel mitochondrial gene (orf522) located 3' to the atpA gene. The dicistronic atpA-orf522 transcripts are preferentially destabilized in male florets of 'restored to fertility' plants as compared with sterile plants. In this report, we show that atpA-orf522 transcripts may be polyadenylated in vivo at their 3' termini and that a tissue-specific increase in the level of polyadenylated atpA-orf522 transcripts correlates with the tissue-specific instability of atpA-orf522 mRNAs in male florets of the restored hybrid plants. In addition, we have identified two distinct ribonuclease activities in sunflower mitochondria, one of which preferentially degrades polyadenylated as compared with non-polyadenylated RNA substrates corresponding to the 3' UTR of atpA-orf522 transcripts. These in vivo and in vitro results show that polyadenylation is involved in the degradation pathway of the mitochondrial atpA-orf522 transcripts and that polyadenylation can be developmentally regulated by a nuclear gene(s) upon restoration of fertility.

Degradation of mRNAs is usually initiated by deadenylation, the shortening of long poly(A) tails to oligo(A) tails of 12-15 As. Deadenylation leads to decapping and to subsequent 5' to 3' degradation by XRN proteins, or alternatively 3' to 5' degradation by the exosome. Decapping can also be induced by uridylation as shown for the non-polyadenylated histone mRNAs in humans and for several mRNAs in Schizosaccharomyces pombe and Aspergillus nidulans. Here we report a novel role for uridylation in preventing 3' trimming of oligoadenylated mRNAs in Arabidopsis. We show that oligo(A)-tailed mRNAs are uridylated by the cytosolic UTP:RNA uridylyltransferase URT1 and that URT1 has no major impact on mRNA degradation rates. However, in absence of uridylation, oligo(A) tails are trimmed, indicating that uridylation protects oligoadenylated mRNAs from 3' ribonucleolytic attacks. This conclusion is further supported by an increase in 3' truncated transcripts detected in urt1 mutants. We propose that preventing 3' trimming of oligo(A)-tailed mRNAs by uridylation participates in establishing the 5' to 3' directionality of mRNA degradation. Importantly, uridylation prevents 3' shortening of mRNAs associated with polysomes, suggesting that a key biological function of uridylation is to confer 5' to 3' polarity in case of co-translational mRNA decay.

Plants infected by citrus tristeza virus (CTV) contain, in addition to the 2000 nm full-length thread-like virions, a heterogeneous population of smaller particles. The CTV particles and RNA extracts from purified CTV preparations were fractionated by sucrose gradient centrifugation and the RNA molecules from different fractions translated in a reticulocyte translation system. Fractions containing predominantly an RNA band of approximately 3.2 kb directed the synthesis of CTV coat protein (CP), which in SDS-PAGE had an estimated molecular mass of 28 kDa. Three additional polypeptides, with estimated sizes of 21 kDa, 23 kDa and 27 kDa, were translated from a range of RNA molecules smaller than 3.2 kb. Hybridization with cDNA to the CP gene (CTV-CPG) and with a 350 base clone complementary to the 3′ and 5′ termini of the genes for CTV p20 and p23.5, respectively, indicated that preparations of CTV particles contain, in addition to the genomic (20 kb) RNA, two sub-genomic RNA molecules of 3.2 kb and 2.4 kb and probably also two smaller molecules of 1.6 kb and 0.9 kb. Only the 3.2 kb RNA, its corresponding dsRNA molecule and populations of larger RNAs, including the 20 kb genomic RNA, hybridized with a CTV-CPG probe, thus conflicting with our previous assignment of the CTV-CPG to the 0.8 kbp dsRNA. Based on these results we propose that distinct populations of CTV particles encapsidate smaller RNAs which were formed as a nested set of subgenomic RNAs. Sequence analysis of 2540 nucleotides downstream to CTV-CPG of strain VT revealed four open reading frames (ORFs) potentially encoding, in the 5′ to 3′ direction, 18 kDa (p18), 13 kDa (p13), 20 kDa (p20) and 23.5 kDa (p23.5) proteins. The CTV-VT ORFs showed variable but usually close levels of homology with the corresponding ORFs of CTV-T36 from Florida.

The exosome is a conserved protein complex that is responsible for essential 3'→5' RNA degradation in both the nucleus and the cytosol. It is composed of a nine-subunit core complex to which co-factors confer both RNA substrate recognition and ribonucleolytic activities. Very few exosome co-factors have been identified in plants. Here, we have characterized a putative RNA helicase, AtMTR4, that is involved in the degradation of several nucleolar exosome substrates in Arabidopsis thaliana. We show that AtMTR4, rather than its closely related protein HEN2, is required for proper rRNA biogenesis in Arabidopsis. AtMTR4 is mostly localized in the nucleolus, a subcellular compartmentalization that is shared with another exosome co-factor, RRP6L2. AtMTR4 and RRP6L2 cooperate in several steps of rRNA maturation and surveillance, such as processing the 5.8S rRNA and removal of rRNA maturation by-products. Interestingly, degradation of the Arabidopsis 5' external transcribed spacer (5' ETS) requires cooperation of both the 5'→3' and 3'→5' exoribonucleolytic pathways. Accumulating AtMTR4 targets give rise to illegitimate small RNAs; however, these do not affect rRNA metabolism or contribute to the phenotype of mtr4 mutants. Plants lacking AtMTR4 are viable but show several developmental defects, including aberrant vein patterning and pointed first leaves. The mtr4 phenotype resembles that of several ribosomal protein and nucleolin mutants, and may be explained by delayed ribosome biogenesis, as we observed a reduced rate of rRNA accumulation in mtr4 mutants. Taken together, these data link AtMTR4 with rRNA biogenesis and development in Arabidopsis.

One of the most versatile RNA degradation machines in eukaryotes is the 3'-5' RNA exosome. It consists of nine conserved subunits forming the core complex, which associates with active ribonucleases, RNA binding proteins, helicases and additional co-factors. While yeast and human exosome core complexes are catalytically inactive, the plant core complex has probably retained a phosphorolytic activity. Intriguingly, the down-regulation of individual subunits of the plant core complex in Arabidopsis mutants led to distinct developmental defects, suggesting an unequal contribution of the core subunits to the in vivo activities of the plant exosome complex. In addition, some of the plant core subunits as well as some associated factors are encoded by duplicated genes, which may have both overlapping and specific functions. Together, these results suggest an unique and complex organisation of exosome-mediated RNA degradation processes in plants. This chapter reviews our current knowledge of plant exosomes and discusses the impact of 3'-5' RNA degradation on the posttranscriptional control of plant genome expression.

One of the most versatile RNA degradation machines in eukaryotes is the 3′–5′ RNA exosome. It consists of nine conserved subunits forming the core complex, which associates with active ribonucleases, RNA binding proteins, helicases and additional co-factors. While yeast and human exosome core complexes are catalytically inactive, the plant core complex has probably retained a phosphorolytic activity. Intriguingly, the down-regulation of individual subunits of the plant core complex in Arabidopsis mutants led to distinct developmental defects, suggesting an unequal contribution of the core subunits to the in vivo activities of the plant exosome complex. In addition, some of the plant core subunits as well as some associated factors are encoded by duplicated genes, which may have both overlapping and specific functions. Together, these results suggest an unique and complex organisation of exosome-mediated RNA degradation processes in plants. This chapter reviews our current knowledge of plant exosomes and discusses the impact of 3′–5′ RNA degradation on the posttranscriptional control of plant genome expression.

In plant mitochondria, polyadenylation-mediated RNA degradation is involved in several key aspects of genome expression, including RNA maturation, RNA turnover, and RNA surveillance. We describe here a combination of in vivo, in vitro, and in organello methods that have been developed or optimized to characterize this RNA degradation pathway. These approaches include several PCR-based methods designed to identify polyadenylated RNA substrates, as well as in vitro and in organello systems, to study functional aspects of the RNA degradation processes. Taken together, identification of RNA substrates combined with information from degradation assays are invaluable tools to dissect the mechanisms and roles of RNA degradation in plant mitochondrial genome expression.

Polyadenylation is a multifunctional post-transcriptional modification that is best known for stabilizing eukaryotic mRNAs and promoting their translation. However, the primordial role of polyadenylation is to target RNAs for degradation by 3' to 5' exoribonucleases. Polyadenylation-assisted RNA degradation contributes to post-transcriptional control in the three genetic compartments of a plant cell: the nucleus, the chloroplast and the mitochondrion. Here, we review the current knowledge of this RNA degradation pathway in these compartments, highlighting recent results that emphasize the crucial role of polyadenylation-assisted RNA degradation in plant genome expression. We also discuss other possible roles of polyadenylation and its sister process polyuridylation.

Yeast Rrp6p and its human counterpart, PM/Scl100, are exosome-associated proteins involved in the degradation of aberrant transcripts and processing of precursors to stable RNAs, such as the 5.8S rRNA, snRNAs, and snoRNAs. The activity of yeast Rrp6p is stimulated by the polyadenylation of its RNA substrates. We identified three RRP6-like proteins in Arabidopsis thaliana: AtRRP6L3 is restricted to the cytoplasm, whereas AtRRP6L1 and -2 have different intranuclear localizations. Both nuclear RRP6L proteins are functional, since AtRRP6L1 complements the temperature-sensitive phenotype of a yeast rrp6Delta strain and mutation of AtRRP6L2 leads to accumulation of an rRNA maturation by-product. This by-product corresponds to the excised 5' part of the 18S-5.8S-25S rRNA precursor and accumulates as a polyadenylated transcript, suggesting that RRP6L2 is involved in poly(A)-mediated RNA degradation in plant nuclei. Interestingly, the rRNA maturation by-product is a substrate of AtRRP6L2 but not of AtRRP6L1. This result and the distinctive subcellular distribution of AtRRP6L1 to -3 indicate a specialization of RRP6-like proteins in Arabidopsis.

In plant mitochondria, polyadenylation‐mediated RNA degradation is involved in several key aspects of genome expression, including RNA maturation, RNA turnover, and RNA surveillance. We describe here a combination of in vivo, in vitro, and in organello methods that have been developed or optimized to characterize this RNA degradation pathway. These approaches include several PCR‐based methods designed to identify polyadenylated RNA substrates, as well as in vitro and in organello systems, to study functional aspects of the RNA degradation processes. Taken together, identification of RNA substrates combined with information from degradation assays are invaluable tools to dissect the mechanisms and roles of RNA degradation in plant mitochondrial genome expression.

Arabidopsis thaliana chloroplasts contain at least two 3' to 5' exoribonucleases, polynucleotide phosphorylase (PNPase) and an RNase R homolog (RNR1). PNPase has been implicated in both mRNA and 23S rRNA 3' processing. However, the observed maturation defects do not affect chloroplast translation, suggesting that the overall role of PNPase in maturation of chloroplast rRNA is not essential. Here, we show that this role can be largely ascribed to RNR1, for which homozygous mutants germinate only on sucrose-containing media, and have white cotyledons and pale green rosette leaves. Accumulation of chloroplast-encoded mRNAs and tRNAs is unaffected in such mutants, suggesting that RNR1 activity is either unnecessary or redundant for their processing and turnover. However, accumulation of several chloroplast rRNA species is severely affected. High-resolution RNA gel blot analysis, and mapping of 5' and 3' ends, revealed that RNR1 is involved in the maturation of 23S, 16S and 5S rRNAs. The 3' extensions of the accumulating 5S rRNA precursors can be efficiently removed in vitro by purified RNR1, consistent with this view. Our data suggest that decreased accumulation of mature chloroplast ribosomal RNAs leads to a reduction in the number of translating ribosomes, ultimately compromising chloroplast protein abundance and thus plant growth and development.

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.

Gene expression is regulated at many stages not merely at the level of transcription. Among the important post-transcriptional processes, RNA turnover has a crucial role. The stability of mRNA in the cytosol of eukarya is increased by the addition of a 3' poly(A) extension. By contrast, this process mediates rapid RNA decay in prokarya. How is mRNA turnover regulated in mitochondria? Their monophyletic, alpha-proteobacterial origin predicts that polyadenylation will induce rapid decay by nucleases and associated factors that are similar to their bacterial ancestors. In this article, however, we report that the regulation of mitochondrial mRNA turnover in diverse species is surprisingly different.

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.