Search

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.

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.

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.

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.

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

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.

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.

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.

We have analysed the expression of heat shock protein 70 (HSP70) and heat shock factor (HSF) gene during maize pollen development, HSFs being the transcriptional activators of hsp genes. In order to eliminate the sporophytic tissues of anthers, we have isolated homogeneous cell populations corresponding to five stages of maize pollen development from microspores to mature pollen. We show that in the absence of heat stress, hsp70 genes are highly expressed late-bicellular pollen as compared to other stages. HSP70 transcripts are significantly accumulated in response to a heat shock at the late microspore stage but to a much lower extent than in vegetative tissues. The latest stages of pollen development, i.e. mid-tricellular and mature pollen, do not exhibit heat-induced accumulation of HSP70 transcripts. Therefore, we analysed the expression of hsf genes throughout pollen development. We demonstrate that at least three hsf genes are expressed in maize and that transcripts corresponding to one hsf gene, whose expression is independent of temperature in somatic as well as in microgametophytic tissues, are present at similar levels throughout pollen development. In addition, we show that the expression of the two other hsf genes is heat-inducible in maize vegetative tissues and is not significantly increased after heat shock at any stage of pollen development. These results indicate that the loss of hsp gene expression at late stages of pollen development is not due to a modification of hsf gene expression at the mRNA level and that hsf gene expression is differentially regulated in vegetative and microgametophytic tissues.We have analysed the expression of heat shock protein 70 (HSP70) and heat shock factor (HSF) gene during maize pollen development, HSFs being the transcriptional activators of hsp genes. In order to eliminate the sporophytic tissues of anthers, we have isolated homogeneous cell populations corresponding to five stages of maize pollen development from microspores to mature pollen. We show that in the absence of heat stress, hsp70 genes are highly expressed late-bicellular pollen as compared to other stages. HSP70 transcripts are significantly accumulated in response to a heat shock at the late microspore stage but to a much lower extent than in vegetative tissues. The latest stages of pollen development, i.e. mid-tricellular and mature pollen, do not exhibit heat-induced accumulation of HSP70 transcripts. Therefore, we analysed the expression of hsf genes throughout pollen development. We demonstrate that at least three hsf genes are expressed in maize and that transcripts corresponding to one hsf gene, whose expression is independent of temperature in somatic as well as in microgametophytic tissues, are present at similar levels throughout pollen development. In addition, we show that the expression of the two other hsf genes is heat-inducible in maize vegetative tissues and is not significantly increased after heat shock at any stage of pollen development. These results indicate that the loss of hsp gene expression at late stages of pollen development is not due to a modification of hsf gene expression at the mRNA level and that hsf gene expression is differentially regulated in vegetative and microgametophytic tissues.

A large-scale purification procedure was developed to isolate the five subunits of F1-ATPase from pig heart mitochondria. The previously described procedure (Williams, N. and Pedersen, P.L. (1986) Methods Enzymol. 126, 484-489) to dissociate the rat liver F1-ATPase by cold treatment followed by warming at 37 degrees C has been adapted for the pig heart enzyme. Removal of endogenous nucleotides from that enzyme before dissociation led to the efficient separation of the alpha and gamma subunits from beta, delta and epsilon subunits. The beta subunit was purified in the hundred-milligram range by anion-exchange chromatography in the absence of any denaturing agent. This subunit was free from any bound nucleotide and almost no ATPase and adenylate kinase-like activities were detected. The delta and epsilon subunits were purified by reversed-phase chromatography (RP-HPLC) in the milligram range. As recently reported (Penin, F., Deleage, G., Gagliardi, D., Roux, B. and Gautheron, D.C. (1990) Biochemistry 29, 9358-9364), these purified subunits kept biophysical features of folded proteins and their ability to reconstitute the tight delta epsilon complex. The alpha and gamma subunits remained poorly soluble and required dissociation by 8 M guanidinium chloride prior to their purification by RP-HPLC. In addition, characterizations of the five subunits by IEF and SDS-polyacrylamide gel electrophoresis are reported, as well as ultraviolet spectra and solubility properties of the beta, delta and epsilon subunits.A large-scale purification procedure was developed to isolate the five subunits of F1-ATPase from pig heart mitochondria. The previously described procedure (Williams, N. and Pedersen, P.L. (1986) Methods Enzymol. 126, 484-489) to dissociate the rat liver F1-ATPase by cold treatment followed by warming at 37 degrees C has been adapted for the pig heart enzyme. Removal of endogenous nucleotides from that enzyme before dissociation led to the efficient separation of the alpha and gamma subunits from beta, delta and epsilon subunits. The beta subunit was purified in the hundred-milligram range by anion-exchange chromatography in the absence of any denaturing agent. This subunit was free from any bound nucleotide and almost no ATPase and adenylate kinase-like activities were detected. The delta and epsilon subunits were purified by reversed-phase chromatography (RP-HPLC) in the milligram range. As recently reported (Penin, F., Deleage, G., Gagliardi, D., Roux, B. and Gautheron, D.C. (1990) Biochemistry 29, 9358-9364), these purified subunits kept biophysical features of folded proteins and their ability to reconstitute the tight delta epsilon complex. The alpha and gamma subunits remained poorly soluble and required dissociation by 8 M guanidinium chloride prior to their purification by RP-HPLC. In addition, characterizations of the five subunits by IEF and SDS-polyacrylamide gel electrophoresis are reported, as well as ultraviolet spectra and solubility properties of the beta, delta and epsilon subunits.

RNA uridylation is a post-transcriptional modification of 3' ends of coding and non-coding RNAs. The best known function of uridylation is to stimulate RNA degradation, but the underlying molecular mechanisms are poorly understood. In this work, I investigate the functions of URT1, the main terminal uridylyltransferase (TUTase) that uridylates messenger RNA (mRNA) in Arabidopsis thaliana. My results indicate a dual role of uridylation in mRNA turnover. First, the addition of terminal uridines by URT1 directly slows down deadenylation and leads to the accumulation of mRNAs with longer polyA tails. Second, URT1 promotes the degradation of deadenylated mRNAs with tails of 10-20 nucleotides, likely by recruiting the decapping activator DCP5 via the M1 motif that is present in the N-terminal intrinsically disordered region of URT1. Both effects are essential to favour the 5’-3’ polarity of mRNA degradation and to prevent the deleterious accumulation of excessively deadenylated and 3' truncated mRNAs., L’uridylation des ARN est une modification post-transcriptionnelle qui consiste en l’ajout d’uridines aux extrémités 3’ des ARN codants et non-codants. La fonction principale décrite pour l’uridylation est la stimulation de la dégradation des ARN cibles. Cependant, les processus moléculaires responsables de cette stimulation sont encore peu connus. Mes travaux de thèse ont eu pour but la caractérisation de la fonction moléculaire de URT1, la principale uridylyltransferase terminale qui uridyle les ARN messagers (ARNm) chez Arabidopsis thaliana. Mes résultats démontrent que URT1 module le profil de polyadenylation des ARNm de deux manières. Premièrement, l’ajout d’uridines en 3‘ des ARNm par URT1 freine la déadénylation et entraîne l’accumulation de queues polyadénylées (polyA) plus longues. Deuxièmement, URT1 est impliqué dans la dégradation d’ARNm uridylés ayant une queue polyA courte de 10-20 nucléotides. Un motif peptidique M1 dans la région N-terminale de URT1 est impliqué dans la dégradation de cette population d’ARNm. Ce motif recrute DCP5, un activateur de l’élimination de la coiffe, qui pourrait stimuler l’élimination de la coiffe et favoriser la dégradation de 5’-3’ de ces ARN cibles. Ainsi, les fonctions d’URT1 sont essentielles pour favoriser la dégradation de 5’-3’ et prévenir l’accumulation délétère d’ARNm excessivement déadenylés et tronqués en 3’.

RNA degradation is an essential step of gene regulation and is critical for development and survival of organisms. Uridylation of mRNAs triggers their degradation and is conserved in most eukaryotes. My PhD thesis is focused on the characterization of URT1, a TUTase responsible for the uridylation of mRNAs in Arabidopsis. I have identified a chain of interactions linking URT1 to factors involved in translation inhibition. Those interactions are likely conserved in all land plants. In addition, I show by 3’ RACE-seq, a method using high throughput sequencing to characterize the 3’ extremities of candidate mRNAs, that URT1 expression levels can control the distribution profiles of uridylated and non-uridylated poly(A) tails. Interestingly, the modification of these distribution profiles is associated to the repression of the expression of a reporter gene, without affecting mRNA steady-state levels. My results contribute to a better understanding of the molecular roles of mRNA uridylation in Arabidopsis and open new perspectives related to the diversity of the roles of uridylation in the metabolism of mRNAs.; La dégradation des ARNm participe à la régulation de l’expression des gènes, régulation essentielle au développement et à la survie des organismes. L’uridylation des ARNm favorise leur dégradation et est un processus conservé chez la plupart des eucaryotes. Mes travaux de thèse ont porté sur la caractérisation de URT1, une TUTase responsable de l’uridylation des ARNm chez Arabidopsis. J’ai notamment établi une chaine d’interactions entre URT1 et des facteurs impliqués dans l’inhibition de la traduction. Ces interactions sont probablement conservées dans l’ensemble des plantes terrestres. De plus, je montre grâce à une méthode d’analyse par séquençage à haut-débit des extrémités 3’ d’ARNm candidats que le niveau d’expression de URT1 ou de différentes versions mutées ou tronquées peut contrôler les profils de distributions de taille des queues poly(A) uridylées et non-uridylées. De manière très intéressante, la modification de ces profils de distributions est associée à la répression de l’expression d’un gène rapporteur, sans modification de l’accumulation des ARNm correspondants. Mes résultats contribuent à une meilleure compréhension des rôles moléculaires de l’uridylation des ARNm chez Arabidopsis et ouvrent des perspectives nouvelles quant à la diversité des rôles de l’uridylation dans le métabolisme des ARNm.

The eukaryotic RNA exosome complex is the main 3’-5’ degradation machinery that plays an essential role in RNA decay, quality control and maturation. The exosome core complex (EXO9) is catalytically inert in yeast and humans, and therefore relies on the catalytic activity of associated RNases, Rrp6 and Rrp44. In this study I demonstrated that EXO9 is catalytically active in Arabidopsis. EXO9’s activity is phosphate-dependent, releases nucleoside diphosphates and is reversible, meeting all criteria of a phosphorolytic activity. Importantly, EXO9’s in vivo substrates include the archetypical exosome substrates, rRNA maturation by-products and 5.8S rRNA precursors. My data show that AtRRP44, EXO9 and AtRRP6L2 sequentially cooperate for the processing of 5.8S rRNA. This work sets a basis for studies aiming at further understanding the biological functions of EXO9’s phosphorolytic activity in a eukaryotic organism.; L’exosome joue un rôle fondamental dans la dégradation de 3’ en 5’ et la maturation des ARNs chez les eucaryotes. Le "coeur" de l’exosome est composé de 9 sous-unités (EXO9). EXO9 est catalytiquement inactif chez l’homme et la levure, et est associé à deux RNases, Rrp6 et Rrp44, responsables de l’activité exonucléolytique de l’exosome. Mes travaux de thèse démontrent que chez Arabidopsis, le coeur de l’exosome EXO9 possède une activité catalytique intrinsèque. Cette activité est dépendante de la présence de phosphate, produit des nucléosides diphosphates et est réversible. Elle possède de ce fait toutes les caractéristiques d’une activité phosphorolytique. L’activité d’EXO9 est impliquée dans l’élimination de sous-produits de la maturation des ARNr et dans la maturation de l’ARNr 5.8S, deux fonctions typiques de l’exosome. Mes travaux révèlent également que AtRRP44, EXO9 et AtRRP6L2 coopèrent de manière séquentielle pour la maturation de l’ARN 5.8S. Mes travaux de thèse constituent la base de travaux futurs visant à comprendre les rôles de l’activité phosphorolytique de l’exosome chez un organisme eucaryote.

RNA degradation is an essential mechanism for the regulation of genome expression. The importance of uridylation for RNA degradation is just emerging. This thesis presents the study of URT1 (UTP :RNA Uridylyltransferase 1) and its role in RNA degradation in Arabidopsis thaliana. URT1 is an uridylyltransferase intrinsically and strictly specific for UTP and is distributive for the first nucleotides added. URT1 uridylates mRNA in vivo after a deadenylation step. This uridylation protects mRNA’s3’ end from further attacks and polarise degradation in the 5’ to 3’ direction. This protection of 3’ ends by uridylation and its conferred polarity of 5’ to 3’ degradation are also detected in polysomes. Uridylation is therefore likely important in case of cotranslational degradation of mRNAs. A region in URT1’s N terminal region predicted to be intrinsically disorganised is required for addressing URT1 to processing bodies. However, following heat shock, the nucleotidyltransferase domain present in the C terminal region of URT1 is sufficient to address URT1 to both processing bodies and stress granules, This work contributes to a better understanding of the mechanisms and roles of uridylation in RNA degradation in Arabidopsis thaliana. These results also open perspectives for studying other functions of uridylation such as translation inhibition.; La dégradation des ARN est un mécanisme essentiel à la régulation de l’expression des génomes. L’importance de l’uridylation dans les mécanismes de dégradation des ARN commence juste à être appréciée. Cette thèse présente l’étudede l’UTP :RNA Uridylyltransferase 1 (URT1) et de son rôle dans la dégradation des ARN chez Arabidopsis thaliana. L’étude des propriétés catalytiques de URT1 montre que cette uridylyltransférase est intrinsèquement spécifique des UTP et distributive pour les premières uridines ajoutées. URT1 est responsable in vivo de l’uridylation des ARNm après une étape de déadénylation, protégeant leur extrémité 3’ et polarisant la dégradation de 5’ en 3’. URT1 est localisée dans le cytosol au niveau des granules de stress et des processing bodies. Le mécanisme d’adressage de URT1 dans les processing bodies implique une partie de la région N terminale prédite comme intrinsèquement désorganisée, alors que le domainenucléotidyltransférase C terminal semble suffisant pour permettre l’adressage de URT1 au niveau des processing bodies et granules de stress en réponse à un stress thermique. Ces travaux de thèse ont permis de mieux comprendre les mécanismes et les rôles de l’uridylation dans la dégradation des ARNm chez Arabidopsis. Ils ouvrent des perspectives dans l’étude d’autres fonctions de l’uridylation comme l’inhibition de la traduction.

The work presented in this manuscript defines a new role of mRNA uridylation, using Arabidopsis as a model organism. RNA uridylation is catalyzed by RNA nucleotidyltransferases belonging to the non canonical poly(A) polymerase (ncPAP) family. Among the 14 genes encoding ncPAPs in Arabidopsis, we identified a terminal uridylyl transferase, TUT1, responsible for mRNA uridylation. Our results show that mRNAs are uridylated by TUT1 after a deadenylation step. Uridylation doesn’t modify mRNA degradation rates but is essential for deadenylated mRNA 3’ end protection against 3’- 5’ exoribonucleolytic attacks and to prevent 3’ truncated aberrant mRNA formation. Interestingly, this protection by uridylation is detected in polysomes. One biological function of mRNA uridylation is to establish a 5’-3’ mRNA degradation polarity that could be essential in the case of cotranslational mRNA decay.; Le travail présenté dans ce manuscrit a permis de définir un nouveau rôle de l’uridylation des ARNm en utilisant Arabidopsis comme organisme d’étude. L’uridylation des ARN est catalysée par des ARN nucléotidyltransférases de la famille des poly(A) polymérases non canoniques ou ncPAP. Parmi les 14 gènes codant pour des ncPAP chez Arabidopsis, nous avons identifié une terminale uridylyl transférase, TUT1, responsable de l’uridylation des ARNm. Nos résultats montrent que TUT1 uridyleles ARNm après une étape de déadénylation. Cette uridylation ne modifie pas la vitesse de dégradation des ARNm mais est essentielle pour prévenir l’attaque des extrémités 3’ des ARNm déadénylés par des activités 3’-5’ exoribonucléasiques et la formation de transcrits aberrants tronqués en 3’. De manière intéressante, cette protection par l’uridylation peut être détectée au niveau des polysomes. Une des fonctions biologiques de l’uridylation des ARNm consiste à établir une polarité de 5’ en 3’ de la dégradation des ARNm. Cette polarité pourrait être essentielle dans le cas d’une dégradation des ARNm en cours de traduction.