Les ARNs interagissant avec Piwi (ARNpi ou piRNA en anglais pour Piwi-interacting RNAs) interagissent avec les protéines de la branche PIWI de la famille des Argonautes. Ils participent à la répression des transposons dans la ligne germinale. Les ARNpi sont produits à partir de deux mécanismes: la biogenèse primaire et le cycle d'amplification Ping-Pong. Plusieurs questions fondamentales sur la biogenèse et la fonction des piRNAs sont encore ouvertes. Le travail de ce mémoire est concentré sur deux caractéristiques au niveau de la séquence dans les populations des ARNpi, le U1-biais et le biais de brin, dont les origines restent mystérieuses. Nos analyses ont révélé de nouvelles fonctions au protéines Piwi, dont les domaines MID et PIWI jouent un rôle essentiel dans la définition des deux biais. La structure cristalline du domaine MID de MIWI montre que l'uridine peut être favorisé comme premier nucléotide au niveau de l'extrémité 5' des ARNpi. De la même manière, nos résultats obtenus grâce aux études in vivo des protéines Piwi de Bombyx mori démontrent que le module MID-PIWI est essentiel pour déterminer le bias de brin des ARNpi s'associant avec les protéines Piwi. De plus, nous avons confirmé le rôle de la méthylation de la région N-terminale des protéines Piwi pour leur localisation dans la cellule. Ensemble, ces résultats offrent de nouveaux détails pour la compréhension de la biogenèse des ARNpi.Les protéines Piwi ne sont pas les seuls composants dans la voie des ARNpi. Des tests génétiques ainsi que des analyses biochimiques ont identifié plusieurs éléments impliqués dans la voie des ARNpi, cependant leurs fonctions restent obscures. J'ai étudié la protéine Vreteno, contenant des domaines Tudor, qui est impliquée dans le processus de biogenèse primaire des ARNpi. En utilisant la lignée cellulaire BmN4, nous avons identifié in vivo un complexe contenant Vreteno, des longues molécules d'ARN antisens, pouvant representer les précurseurs des ARNpi associés avec Siwi, ainsi que d'autres composants de la voie ARNpi, comme Ago3, BmTdrd12 et Spindle-E. De plus amples analyses sont nécessaires pour comprendre les fonctions de Vreteno dans la voie des ARNpi.Enfin, nous avons également étudié le rôle de l'hélicase à ARN Vasa dans la voie des piRNAs. Nos résultats démontrent que Vasa assemble un complexe régulé par l'ATP sur le ARN messager des transposons, afin de produire de nouveaux ARNpi secondaires avec orientation sens. En étudiant les différentes étapes de la voie des ARNpi, nos analyses ont fourni des informations importantes pour la compréhension de la biogénèse des ARNpi. / Piwi-interacting RNAs (piRNAs) associate to members of the PIWI clade of Argonaute proteins and are responsible for silencing of transposable elements in animal germ lines. piRNAs are produced through two biogenesis pathways, known as primary processing and Ping-Pong amplification cycle. There are many fundamental questions regarding piRNA biogenesis and function that are unsolved. In this thesis I have been focusing on the presence of two sequences features in piRNA populations, the U1-bias and the strand-bias, whose determination is not understood. Our analyses have revealed new features of the Piwi proteins, whose MID and PIWI domains play an essential role in the definition of both piRNA biases. The crystal structure of the MID domain of MIWI shows that uridine can be favored as the first nucleotide at the 5' end, while our in vivo results on Bombyx mori Piwi proteins demonstrate that the MID-PIWI module is essential to determine the strand bias of piRNAs that associate to the Piwi protein. Moreover we have confirmed the role of the methylation status of the N-terminus of Piwi protein for determining their localization. Altogether these findings provide new insights in the understanding of piRNA biogenesis.Piwi proteins are not the only components acting in the piRNA pathway. Genetic screenings and biochemical analyses have identified several other factors, which have been involved in the piRNA pathway, but whose functions remain elusive. I have focused on the Tudor domain-containing protein Vreteno, which has been involved in the primary biogenesis pathway. Using BmN4 cells, we have identified an in vivo complex of Vreteno, containing long antisense RNA molecules, which might represent the precursor of Siwi-piRNAs, and other piRNA known factors, like Ago3, BmTdrd12 and Spindle-E. Further analyses are required for the understanding of Vreteno functions in the piRNA pathway.Finally, we have also investigated the role of the RNA helicase Vasa in the piRNA pathway. Our results show that Vasa assembles an ATP-gated Ping-pong complex on transposon mRNAs to generate new sense-oriented secondary piRNAs. By looking at different step in the piRNA pathway our analyses have provided important insights for the understanding of the piRNA biogenesis.

In C. elegans, the germline is a tightly regulated tissue where silencing pathways regulate genes, allowing expression of “self” while silencing “non-self.” Doublestranded RNAs (dsRNAs), short interfering RNAs (siRNAs), and piwi-associated RNAs (piRNAs) can transmit this regulation across generations via transgenerational epigenetic inheritance (TEI) mechanisms (Bošković and Rando, 2018). Analogously, some pathways can counteract gene silencing to allow sustained expression in the germline. One such example is a non-coding DNA structure called Periodic An/Tn clusters that can prevent the silencing of transgenes in the germline (Frøkjær-Jensen et al., 2016). In this thesis, I developed a novel piRNA-based tool called piRNA interference (piRNAi), where target-specific short “guide” piRNAs (sg-piRNAs) can robustly silence endogenous genes and transgenes. I have used piRNAi to understand the rules for licensing gene expression and transgenerational epigenetic inheritance in the C. elegans germline. Initially, I describe design rules for generating transgenes with PATC-rich introns that resist germline silencing and are robustly expressed from extrachromosomal arrays. PATC-rich transgenes showed more accurate gene expression patterns and did not prevent germline regulation by 3’ untranslated regions (3’ UTRs). Next, I developed the piRNAi technique to understand the role of PATCs in licensing transgene expression and the rules for how endogenous genes can be targeted for piRNA-mediated silencing and TEI. I demonstrate that a PATC-rich gfp transgene and endogenous genes are not resistant to piRNA-mediated silencing. Finally, I used piRNAi to define rules for TEI: 1. I identified two new endogenous targets for TEI (him-5 and him-8) that can inherit silencing for four and six generations respectively, after transient exposure to sg-piRNAs. 2. I demonstrate that an endogenous gene (him-5) can be semi-permanently silenced in the absence of the piRNA/PRG-1 pathway. 3. The duration of TEI was significantly shortened in a transgene that contained PATC-rich introns. Altogether, my thesis shows that an endogenous small RNA pathway can be reprogrammed to silence endogenous genes and transgenes in the germline, which enables novel experimental paradigms for studying inherited and semipermanent silencing.

Les rétrovirus endogènes sont des parasites génétiques qui s’insèrent dans l’ADN génomique. Bien que leurs insertions délétères soient éliminées par la sélection naturelle, ils prolifèrent et sont une source de plasticité génomique. L’étude de l’impact de leur mobilité sur le génome hôte est rendue difficile par le faible taux de transposition de ces éléments, réprimés par des petits ARN appelés piARNs. Nous avons développé une approche génétique permettant d’inactiver ce contrôle et de déterminer l’impact sur le génome de la drosophile, d’une transposition réplicative. Nous avons remarqué la mise en place d’autres mécanismes de répression des rétrovirus endogènes lors de la perte des piARNs pouvant ainsi limiter leur propagation. Nous avons aussi identifié de nouveaux sites d’intégrations des rétrovirus endogènes après un cycle de transposition réplicative. Cependant, le taux de transposition reste faible. Ce projet combinant différentes approches (génétique, séquençage à haut débit et bioinformatique) a permis de démontrer que la voie des piARNs n’est pas cruciale pour le maintien de l’intégrité du génome, et que d’autres mécanismes semblent intervenir afin de maintenir sa stabilité. / Endogenous retrovirsuses are genetic parasites which are inserted in the genomic DNA. Although their deleterious insertions are eliminated by natural selection, they proliferate and are a source of genomic plasticity. The study of the impact of their mobility on the host genome is made difficult by the transposition’s low rate of these elements, suppressed by a class of small RNA, called piRNA. We have developed a genetic approach to inactivate this control and determine the impact on Drosophila’s genome, after one replicative transposition. We noticed the establishment of other endogenous retroviruses repression mechanisms that are awakened after the loss of the piRNA and they are able to limit their spread. We identified new integrations sites of endogenous retrovirus after one replicative transposition. But we noticed that the transposition rate still low. This project combines different approaches (genetics, high-throughput sequencing and bioinformatics) and show the piRNA pathway is not essential to maintain genome integrity but other mechanisms involving small RNA can be implicated in the genome stability.

MicroRNAs are short non-protein coding RNAs which negatively regulate gene expression by guiding the RNA-induced silencing complex to complementary target mRNAs. MicroRNA regulation is implicated in essentially all biological processes, and microRNAs have a prominent role in animal development. Several microRNA families are conserved between deuterostomes and protostomes, however the majority of microRNAs in animal species are a result of continuous de novo gene birth processes throughout natural history. The acquisition of novel microRNAs, and changes in existing microRNAs, has been suggested to play a role in animal evolution. However, the constraints on microRNA emergence, evolution and expression are not well understood. We have studied the interplay of microRNA developmental expression and evolutionary dynamics in model insects displaying different modes of embryogenesis. We first determined the evolutionary origins and rates of change of microRNAs in Drosophila melanogaster and Drosophila virilis, and analysed their temporal expression profiles throughout development by deep sequencing. We found a good correlation between microRNA conservation and abundance at most stages except for the early embryo, where fast-evolving microRNAs are highly expressed. We further showed that the temporal expression of orthologous microRNAs is highly similar between species, and the global microRNA profiles across development display an hourglass-like conservation pattern, previously observed for protein-coding genes. We next extended our analyses to the red flour beetle Tribolium castaneum, which develops via the short germband mode of embryogenesis. This developmental mode is ancestral and widespread in arthropods, yet the microRNA complement of a representative species has not been previously characterized. We find a number of conserved features between Drosophila and Tribolium, including microRNA maternal loading and modifications, and microRNA-mediated targeting of maternally deposited transcripts. We also describe an abundant pool of maternally loaded and zygotically expressed piRNAs, which appear to be turned on in response to active transposons. In contrast to the previously observed piRNA profile in flies, beetle piRNAs are abundant throughout the entire embryogenesis. A majority of the newly identified microRNAs in the flour beetle are expressed during a discrete period in the early blastoderm, and also target maternally provided transcripts. The observation that the early embryos of both Drosophila and Tribolium are uniquely characterized with high levels of novel and dynamically evolving microRNAs suggests a conserved phenomenon where the blastoderm stage is a highly permissive environment for microRNA innovations.

MicroRNAs are small RNA molecules that could possibly play a major role in fertility. In the experiment, spermatozoa were extracted from bovine followed by an extraction of total RNA. Bovine spermatozoa were extracted from two bulls of different fertility, high and low fertility. An expression array was done to compare the expression levels of the microRNAs. It was shown that thousands of microRNAs are present in bovine spermatozoa but only a small amount was significantly expressed. The microRNAs from low fertility bulls were more highly expressed than those in high fertility bulls. A Bioanalyzer gel was used to confirm the results of the microarray data. The microRNAs were present in the bull’s spermatozoa at 25 nucleotides. The functions of the significantly expressed microRNAs are not known but there is a great possibility that their functions affect fertility.

Eukaryotes have developed stringent regulatory mechanisms that control cell division and ensure proper chromosome segregation. Maintaining genome integrity is especially important during meiosis, the specialised cell division programme in the germline that generates haploid gametes. As these cells transmit genetic information to the next generation, the consequences of meiotic errors are not restricted to an organismal level, but can directly impact the fitness of the offspring. Mammals display a high degree of sexual dimorphism in meiosis with regard to the stringency of regulatory mechanisms. This manifests in a relatively high degree of maternally-derived aneuploidies due to weaker checkpoint control in females, whereas more rigorous checkpoints in males frequently perturb fertility. Mouse models of aneuploidy often exhibit complete male sterility and early germ cell arrest, preventing the study of aneuploidy during late and post-meiotic stages in males. In this thesis, we have used the trans-chromosomic mouse model, Tc1, which carries a single copy of human chromosome 21 (HsChr21) and show that, unlike other aneuploid mouse strains, the Tc1 mouse can successfully passage the exogenous human chromosome through male meiosis and generate aneuploid offspring. Our investigations have shown that the presence of the aneuploid human chromosome causes spermatogenic defects due to an arrest at the first meiotic division. Despite this impairment, we found an unexpectedly high number of aneuploid gametes in Tc1 males and the majority of males were able to produce aneuploid offspring, albeit at a lower frequency. Transmission of HsChr21 through the male germline was less efficient compared to female germline transmission, but allowed us to study the impact of male germline-associated chromatin remodelling on the transcriptional deployment of HsChr21 in the offspring. This revealed that, despite fundamentally different developmental dynamics, male- versus female-germline passage result in indistinguishable transcriptional and regulatory phenotypes. An important pathway in the male germline involves the expression of piRNAs, a class of small non-coding RNAs that are commonly found in the germline of animals where they defend cells against transposable elements. Profiling the expression of small RNAs in the Tc1 mouse showed that conserved human piRNA clusters can be successfully transcribed by the mouse piRNA machinery. In addition, we detected Tc1-specific piRNA sequences that were neither present in human nor mouse, mapping to a human-specific repeat element. In line with the previously observed activation of human-specific repeat elements in the Tc1 mouse, this suggests that novel transcripts arising from human repeats can trigger an adaptive piRNA response, thereby demonstrating the plasticity of this pathway to newly invading repeat elements. Transcriptional profiling of spermatogenic cell populations on a single-cell level allowed us to generate an atlas of gene expression over the course of spermatogenesis and dissect meiotic silencing dynamics in the presence of aneuploidy. Transcriptional silencing during meiosis occurs in response to unpaired chromosomes and, in male germ cells, affects the sex chromosomes due to their largely unpaired nature. We found that the presence of HsChr21 has no impact on the silencing of chromosome X, however, the two chromosomes display drastically different silencing patterns with HsChr21 showing a much weaker repression. Taken together, this study revealed a higher than expected tolerance for aneuploidy in the mouse male germline thus allowing the characterisation of meiotic checkpoint mechanisms, the meiotic silencing response to unpaired chromosomes as well as piRNA expression in the presence of an exogenous human chromosome.

Ces dernières années, de nombreuses études transcriptomiques à grande échelle ont clairement mis en évidence que la grande majorité du génome des eucaryotes est transcrite.Ce réseau complexe de transcrits inclus des petits ARN non codants qui interviennent généralement en tant que régulateurs transcriptionnels, post-transcriptionnels et/ou traductionnels de l’expression de certains ARNm cibles spécifiques. Ils sont classés selon leur origine biologique et leur mode d’action. Une catégorie de petits ARN non codants, les Piwi-interacting RNAs (piRNA), maintient l’intégrité du génome dans les tissus reproducteurs des métazoaires en réprimant les éléments transposables endogènes, des séquences ADN capables de se déplacer et de se dupliquer à l’intérieur du génome. Les piRNA sont produits par deux mécanismes : i) La biogenèse primaire à partir de longs ARN simple brin produits par certains loci spécifiques du génome, les clusters de piRNA, des loci énigmatiques, localisés dans les régions hétérochromatiques et composés de fragments d’éléments transposables actifs, ii) La boucle d’amplification appelée ping-pong. Durant ma thèse, j’ai étudié un cluster de piRNA majeurs dans les cellules somatiques des gonades femelles de Drosophila melanogaster, le locus flamenco. Tout d’abord, j’ai mis en évidence que la transcription de flamenco est initiée à partir d’un promoteur contenant une séquence INR et un élément DPE, reconnu par l’ARN polymerase II, et qu’elle nécessite la présence du facteur de transcription Cubitus Interruptus. Ensuite, j’ai montré que le transcrit de flamenco subit de l’épissage alternatif pour générer divers précurseurs ARN qui seront ensuite maturés en piRNA. De plus, j’ai montré que le promoteur de flamenco serait suffisant pour déclencher l’adressage du transcrit vers la voie de maturation des piRNA. Dans un autre axe, je me suis intéressée à l’organisation tridimensionnelle du locus flamenco au sein du noyau en recherchant ses partenaires d’interaction en utilisant la technique de 4C (capture de la conformation des chromosomes). J’ai pu voir que flamenco semble interagir physiquement avec des régions génomiques fortement transcrites en cis. En trans, le locus flamenco interagit majoritairement avec des régions génomiques péricentromériques et avec d’autres clusters de piRNA. Cette disposition tridimensionnelle particulière pourrait être le reflet d’une organisation fonctionnelle.Dans l’ensemble, ces travaux permettent de mieux comprendre l’expression et le fonctionnement du locus flamenco et ouvrent la voie vers de nouvelles recherches prometteuses. / The past few years it has become clear from many transcriptomic studies that most of the eukaryotic genome is pervasively transcribed. This complex network of transcripts include several types of small RNAs classified as non-coding RNAs. The vast majority of small RNA act as transcriptional, posttranscriptional and/or translational regulators, controlling specific target mRNAs involved in various cellular functions. They are classified based on their biogenesis and mode of action. A subclass of small non-coding RNAs, the Piwi-interacting RNAs (piRNA), ensures genomic stability by silencing endogenous transposable elements, endogenous sequence that are able to move and duplicate into the genome, in both germline and somatic gonadal tissues of metazoan. piRNA are produced through two mechanisms, i) The primary processing pathway from long single-stranded precursors produced by some specific loci in the genome, the piRNA clusters, ii) The secondary pathway by the amplification loop called the ping-pong. piRNA clusters are enigmatic loci localized in heterochromatic region and composed of transposable element fragments.During my PhD, I studied a major piRNA cluster in the somatic cells of Drosophila melanogaster female gonads, the flamenco locus. First, I demonstrated that flamenco transcription is initiated from an RNA Polymerase II promoter containing Inr and DPE elements, and requires the transcription factor, Cubitus interruptus. Then, I showed that the flamenco precursor transcript undergoes differential alternative splicing to generate diverse RNA precursors that are processed into piRNA. Moreover, I showed that the flamenco promoter could be sufficient to target transcripts into the piRNA processing pathway. In an other hand, I was interested to the tridimensional nuclear organization of the flamenco locus using the 4c technology. I saw that the flamenco locus interacts physically with strongly transcribed genomic region in cis. In trans, the flamenco interacts with other peri-centromeric genomic regions and with two other piRNA cluster. This particularly three-dimensional positioning could be the reflect of a functional organization.In the main, this work allows to better understand the expression and the mode ofaction of the flamenco locus and paves the way for new promising research.

Les protéines Argonaute sont associées à de petits ARN et participent à la régulation de l'expression des gènes. Les protéines Piwi, sous-famille des protéines Argonaute, sont principalement exprimées dans les lignées germinales. Elles recrutent les piRNA (Piwi-interacting RNA) et assurent la stabilité du génome en inhibant les transposons. Une caractéristique des piRNA est la présence de groupes 2'-O-methyl à l'extrémité 3'. Les microARN et siRNA (small interfering RNA) de plantes, comme les siRNA de Drosophyle portent aussi cette modification qui est catalysée par l'ARN méthyl-tranférase HEN1. Son homologue murin, mHEN1, méthyle in vitro de petits ARN, mais son rôle dans la voie des piRNA n'avait pas encore été envisagé. Mon objectif était de relier mHEN1 à la voie des piRNA. J'ai démontré que mHEN1 interagit directement avec la partie N-ter de MILI mais pas avec les autres protéines Piwi de souris. La partie N-ter de MILI porte des arginines méthylées. J'ai démontré que l'interaction ne dépendait pas de la présence de cette modification, ce qui suggère que mHEN1 intervient avant la modification de MILI. Par imagerie cellulaire j'ai montré la compartimentation de HEN1 et des protéines Piwi dans des granules cytoplasmiques différents. Parallèlement, afin de caractériser les éléments de la voie piRNA, j'ai développé un nouveau modèle d'étude basé sur des embryons de poisson zèbre (Danio rerio). Ainsi, j'ai évalué le rôle de deux protéines interagissant avec les protéines Piwi, TDRD1 (Tudor-domain containing) et l'hélicase MOV10l décrits chez la souris mais pas chez le poisson zèbre. J'ai montré que l'expression de DrTDRD1, spécifique à la lignée germinale, dépend de sa partie 3'UTR. La réduction de l'expression de DrMOV10l, obtenue grâce à l'utilisation de morpholinos, entraîne la dérépression des éléments rétrotransposables des embryons en développement. Cette technique de Knock Down sera utilisée pour identifier de nouveaux éléments de la biogenèse des piRNA. / Argonaute proteins associate with small RNAs to participate in gene regulatory processes. Piwi proteins are a sub clade of Argonaute that are mainly expressed in the germ line. They bind to Piwi-interacting RNAs (piRNAs) and exert functions in genome stability through transposon silencing. One defining feature of piRNAs is the presence of a 2'-O-methyl group on the 3' terminal nucleotide. Plants microRNAs (miRNAs) and small interfering RNAs (siRNAs) as well as Drosophila siRNAs carry a similar modification, which is catalyzed by the RNA methyltransferase HEN1. The mouse homolog, mHEN1, was shown to have methylation activity for RNA substrates in vitro, but its role in the murine piRNA pathway had not been addressed. My aim was to connect mHEN1 to the piRNA pathway. I demonstrated that mHEN1 interacts directly with the N-terminus of MILI but not with the other murine Piwi proteins. The N-terminus of MILI is known to carry methylated arginines, but I show that this interaction is independent of post-translational modification of MILI. Cellular imaging experiments identified compartmentalization of the enzyme and Piwi proteins into distinct cytoplasmic granules. These studies delineated interactions between mHEN1 and piRNA pathway factors and suggest that the enzyme can act prior to the arginine methylation of MILI. In a parallel study, I developed zebrafish (Danio rerio) embryos as a model system for identifying and manipulating piRNA pathway components. To this end, I evaluated the role of the two Piwi-interacting proteins, the Tudor-domain containing protein DrTDRD1 and the putative helicase DrMOV10l described in mouse but not in zebrafish. I showed that the germ line-specific expression of DrTDRD1 is dependent of its 3'UTR. A loss of DrMOV10l by morpholino knock down results in derepression of retrotransposable elements in the developing embryos. This morpholino-based technique I set up will be used to identify new components of the biogenesis of piRNAs.

La spermatogenèse est le processus par lequel les cellules germinales se différencient pour former les spermatozoides. Elle se déroule à l’intérieur des tubes séminifères. Pendant la période embryonnaire, les précurseurs des cellules germinales adultes constituent un pool de cellules appelées cellules germinales primordiales (Primordial Germ Cells, PGCs), qui vont migrer pour aller coloniser les gonades (Durcova-Hills and Capel, 2008; Surani et al., 2008). Au cours de leur migration, les PGCs vont subir une reprogrammation épigénétique de l’ensemble de leur génome, qui leur sera nécessaire pour passer de l’état somatique à l’état de totipotence (Ohinata et al., 2005). Durant cette reprogrammation, l’ADN est massivement démethylé, entrainant l’activation de plusieurs gènes qui sont importants pour le développement des PGCs, mais également l’activation des éléments transposables (ETs) (Hajkova et al., 2008; Sasaki and Matsui, 2008; Surani and Hajkova, 2010). Ces derniers constituent environ 50% du génome des mammifères. Ils sont subdivisés en deux classes et sont connus par leur capacité à être mobilisés dans le génome (Zamudio and Bourc'his, 2010). Cette mobilisation se fait au hasard et constitue ainsi un risque considérable de mutations, qui peuvent provoquer des tumeurs, des pathologies de développement et une infertilité (Zamudio and Bourc'his, 2010). Pour cela, leur expression doit être contrôlée pour maintenir l’intégrité du génome de la lignée germinale. Pour toutes ces raisons, les PGCs ainsi que les cellules germinales en cours de méiose ont développé des stratégies de défenses pour contrôler la mobilisation et l’expression des ETs basées entre autre sur la voie des piwi-interacting RNA (piRNA) (Chuma and Pillai, 2009; Pillai and Chuma, 2012b). Dans le laboratoire du Pr. Stéphane Viville, mes travaux de thèse se sont concentrés sur l’étude d’un gène nommé Tex19 pour Testis Expressed gene chez la souris. Nous avons démontré que ce gène est spécifique des mammifères et est dupliqué chez le rat et la souris en deux paralogues nommés Tex19.1 et Tex19.2. Deux domaines hautement conservés ont été identifiés par alignement multiple des protéines TEX19 et nommés MCP et VPTEL. Ces domaines ne présentent aucune homologie avec des domaines déjà caractérisés, prévenant ainsi toute prédiction de leurs fonctions (Kuntz et al., 2008). L’étude du profil d’expression de Tex19.1 et Tex19.2 a montré que ces deux gènes sont exprimés dans l’ectoderme et les PGCs. Ils sont aussi co-exprimés dans le testicule de l’âge embryonnaire à l’âge adulte. Néanmoins, seul Tex19.1 est exprimé dans les ovaires et le précurseur du placenta appelé cône ectoplacentaire (Celebi et al., 2012). Le knockout (KO) de Tex19.1 provoque une infertilité masculine chez la souris avec un arrêt de la spermatogenèse au stade pachytène, accompagnée d’une surexpression d’un rétrotransposon, MMERVK10C (Ollinger et al., 2008). Récemment, il a été démontré que Tex19.1 joue aussi un rôle dans le développement du placenta (Reichmann et al., 2013). Au cours de mes trois années de thèse, nous avons approfondie l’étude du KO de Tex19.1dans le testicule, les cellules embryonnaires souches (Embryonic Stem Cells, ESCs) et le placenta (Tarabay et al., 2013). Nous avons également étudié le phénotype observé suite au double KO de Tex19.1 et Tex19.2. [...] / We recently characterized two new mammalian specific genes, Tex19.1 and its paralog Tex19.2. Both genes are expressed in pachytene spermatocytes in adult testes. In addition, Tex19.1 is expressed in pluripotent cells (ES, EG, iPS and PGC cells), the inner cell mass of the blastocysts and the placenta. In order to decipher Tex19 functions, we generate three types of knockout (KO): i) KO of Tex19.1 ii) KO of Tex19.2 iii) double KO (DKO) of both genes. All Tex19.1-/- KO animals are growth-retarded and half of them die just after birth. This phenotype is probably linked to placenta defects. Surviving adults Tex19.1-/- KO males display a variable spermatogenesis phenotype, associated with an up-regulation of one endogenous retrovirus, MMERVK10C. Tex19.2 KO mice exhibit a subtle phenotype. Few seminiferous epitheliums are degenerated while the rest appear normal. DKO show a fully penetrant phenotype similar to the most severe Tex19.1-/- phenotype. DKO males exhibit small testes. Despite the presence of spermatogonia and spermatocytes, spermatogenesis is blocked at the pachytene stage. By RNA deep-sequencing on 10 days old DKO and WT testes, prior to histological phenotype, 114 genes are significantly up-regulated and 320 genes significantly down-regulated in the DKO compared to the WT. Gene ontology analyses show that among of these genes, two essential pathways are altered: meiosis and the piRNA pathway. Consistent with that, GST-pulldown and immunoprecipitation experiments demonstrate that MIWI, MILI, MAEL and MVH are partners of TEX19. Considering PIWI proteins function in the silencing of transposable elements through the piRNA pathway, we checked if TEX19 paralogs bind piRNA. By immunoprecipitation using WT and KO testes, we show that both TEX19.1 and TEX19.2 bind small RNA of 30 nucleotides through their VPTEL domain. This study highlights the pivot role of Tex19 paralogs in three essential functions of mammalian life cycle, i.e. pluripotency, placenta-supported in utero growth and fertility. The functional similarities of both paralogs, through the expression control of one endogenous retrovirus and the binding of piRNAs, lead us to propose that Tex19 paralogs are new members of the piRNA pathway.

The discovery of RNA in 1868 by Friedrich Miescher was meant to be the prologue to an exciting new era in Biology full of scientific breakthroughs and accomplishments. Since then, RNAs have been proven to play an indispensable role in biological processes such as coding, decoding, regulation and expression of genes. In particular, the discovery of small non-coding RNAs and especially miRNAs, in C. elegans first and thereafter to almost all animals and plants, started to fill in the puzzle of a complex gene regulatory network present within cells. The aim of this thesis is to shed more light on the features and functionality of small RNAs. In particular, we will focus on the function and biogenesis of miRNAs and piRNAs, across multiple species, by employing advanced computational methods and machine learning. We first introduce a novel method (Chimira) for the identification of miRNAs from sets of animal and plant hairpin precursors along with post-transcriptional terminal modifications that are not encoded by the genome. This method allows the characterisation of the prevalence of miRNA isoforms within different cell types and/or conditions. We have applied Chimira within a larger study that examines the effect of terminal uridylation in RNA degradation in oocytes and cells in either embryonic or adult stage. This study showed that uridylation is the predominant transcriptional regulation mechanism in oocytes while it does not retain the same functionality on mRNAs and miRNAs, both in embryonic and adult cells. We then move on to a large-scale analysis of small RNA-Seq datasets in order to identify potential modification signatures across specific conditions and cell types or tissues in Human and Mouse. We extracted the full modification profiles across 461 samples, unveiling the high prevalence of modification signatures of mainly 1 to 4 nucleotides. Additionally, samples of the same cell type and/or condition tend to cluster together based on their miRNA modification profiles while miRNA gene precursors with close genomic proximity showed a significant degree of co-expression. Finally, we elucidate the determinant factors in strand selection during miRNA biogenesis as well as update the miRBase annotation with corrected miRNA isoform sequences. Next, we introduce a novel computational method (mirnovo) for miRNA prediction from RNA-Seq data with or without a reference genome using machine learning. We demonstrate its efficiency by applying it to multiple datasets, including single cells and RNaseIII deficient samples, supporting previous studies for the existence of non-canonical miRNA biogenesis pathways. Following this, we explore and justify a novel piRNA biogenesis pathway in Mouse which is independent of the MILI enzyme. Finally, we explore the efficiency of CRISPR/Cas9 induced editing of miRNA targets based on the computationally predicted accessibility of the targeted regions in the genome. We have publicly released two web-based novel computational methods and one on-line resource with results regarding miRNA biogenesis and function. All findings presented in this study comprise another step forward within the journey of elucidation of RNA functionality and we believe they will be of benefit to the scientific community.