Candida glabrata est une levure pathogène opportuniste, apparaissant aujourd’hui comme la deuxième cause de candidémie en Europe et en Amérique du Nord. Cette levure présente de nombreuses particularités génomiques telles que la présence de nouveaux domaines structuraux au sein d’ARN non-codants ubiquitaires. Le premier aspect de cette thèse a consisté en l’étude de la sous-unité ARN atypique de la Ribonucléase P nucléaire de C. glabrata. Cet ARN contient trois grand domaines additionnels octroyant au transcrit une taille trois fois plus élevée que la moyenne des sous-unités ARN des RNase P eucaryotiques. Les expériences réalisées ont permis une meilleure compréhension du rôle de ces domaines additionnels et ont démontré la présence inédite de la protéine Rcl1 au sein du complexe de la RNase P. Dans un second temps ce travail de thèse a aussi contribué à l’amélioration des outils d’édition du génome de C. glabrata existants. De nouvelles cassettes intégratives de faible taille et positivement sélectionnables ont été mises au point. Ces éléments présentent toutes les caractéristiques permettant leur utilisation dans la modification du génome de souches sauvages et d’isolats cliniques de C. glabrata. / Candida glabrata is an opportunistic pathogenic yeast, and is today the second causative agent of candidemia in Europe and North America. This yeast has many genomic peculiarities such as the presence of new structural domains within ubiquitous non-coding RNAs. The first aspect of this thesis was the study of the atypical RNA subunit of the nuclear Ribonuclease P of C. glabrata. This RNA contains three large additional domains giving the transcript an overall size more than three times larger than the average eukaryotic RNase P RNA subunits. The experiments performed led to a better understanding of the role of these additional domains and demonstrated for the first time the presence of the Rcl1 protein within the RNase P complex. Secondly, this thesis work also contributed to the improvement of existing genome editing tools in C. glabrata. New small and positively selectable integrative cassettes have been developed. These elements exhibited all the required characteristics for their use in wild-type strains and clinical isolates of C. glabrata.

Orientador: Cesar Martins / Resumo: Os cromossomos B ocorrem em cerca de 2.828 espécies de diferentes táxons, sendo basicamente heterocromáticos e compostos de DNAs repetitivos. Recentemente, análises genômicas em larga escala estão sendo utilizadas para elucidar questões acerca dos cromossomos supranumerários. Os peixes ciclídeos recebem grande interesse científico, uma vez que muitas espécies passaram por um rápido e extenso processo de radiação adaptativa. Em algumas espécies do grupo, como Astatotilapia latifasciata, foi descrita a presença de cromossomos B. Neste trabalho foi caracterizado o perfil de expressão proteico em tecidos específicos na A. latifasciata e realizada análise funcional da presença do cromossomo B nesta espécie de teleósteo, elucidando a influência que este pode acarretar em vias metabólicas específicas. Além disso, esses dados foram integrados com os resultados de RNA-Seq dessa espécie, e construídas sub redes de co-expressão e interação proteína-proteína. Também foi calculada a entropia de Shannon, a qual não apresentou diversidade na expressão dos transcritos em cada biblioteca comparada. Além disso, foi analisada a expressão diferencial de RNAm em cada tecido em relação a presença do cromossomo B e ao sexo. Dentre os transcritos diferencialmente expressos, a análise de enriquecimento funcional apresentou processos relacionados ao ciclo celular, resposta imune e resposta ao estresse. Na maioria dos casos analisados entre a expressão de proteínas, os transcritos up regulated e as sub... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The B chromosomes occur in about 2,828 species of different taxa, being heterochromatic and composed of repetitive DNA.Recently, large-scale genomic analyzes are being used to elucidate questions about supernumerary chromosomes.Cichlid fish are of great scientific interest, since many species have gone through a rapid and extensive process of adaptive radiation.In some species of the group, such as Astatotilapia latifasciata, the presence of B chromosomes was described.In this work, we characterize the profile of protein expression in specific tissues in A. latifasciata and performed a functional analysis of the presence of the B chromosome in this species of teleost, elucidating the influence that it can cause in specific metabolic pathways.In addition, we integrate these data with the RNA-Seq results of this species, and construct sub-networks of co-expression and protein-protein interaction.The Shannon entropy was also calculated, which did not show diversity in the expression of the transcripts in each library. In addition, differential expression analysis was performed on each tissue separately and the relationship between the presence of B chromosome and sex chromosome was analyzed. Among the differentially expressed transcripts, functional enrichment analysis presented processes related to the cell cycle, immune response and stress response. In relation to abundance of proteins, the up regulated transcripts and the sub-networks we identified genes like the Aurora kinas... (Complete abstract click electronic access below) / Doutor

Análises de dados gerados em larga escala revelaram que os transcriptomas são mais extensos e complexos do que inferido previamente. Hoje é evidente que a maioria dos genomas de eucariotos são quase inteiramente transcritos e sob regulação atrelada a estágios de desenvolvimento. O controle da expressão gênica envolve RNAs não codificadores (ncRNAs) regulatórios, inclusive em processos pós-transcricionais. A regulação de expressão gênica em nível pós-transcricional é crucial em diferentes organismos, mas é particularmente central nos tripanossomatídeos. Os parasitos do gênero Leishmania (Ordem Kinetoplastidae, família Trypanosomatidae) provocam doenças infecto-parasitárias conhecidas como leishmanioses e em seu ciclo de vida apresenta-se sob três formas principais de desenvolvimento: promastigotas procíclicos, promastigotas metacíclicos e os amastigotas. Este trabalho teve como objetivo identificar e caracterizar computacionalmente ncRNAs putativos de Leishmania em diferentes estágios de desenvolvimento. A associação de abordagens em larga escala e ferramentas de bioinformática possibilitaram a identificação de 11.376 ncRNAs putativos em L. braziliensis e, em um estudo preliminar, de 37 ncRNAs putativos em L. donovani. Adicionalmente, o transcriptoma de L. braziliensis foi analisado comparativamente entre os três estágios de desenvolvimento do parasito. Em L. donovani, dos 37 ncRNAs putativos identificados, 34 estavam em UTRs (Untranslated Regions) e 3 em regiões intergênicas. Preditores de características específicas de ncRNAs foram utilizados e 32 ncRNAs putativos tiveram pelo menos uma predição positiva. Todos os candidatos estão conservados inteira ou parcialmente em pelo menos 3 espécies de Leishmania. Cinco ncRNAs de L. donovani foram confirmados por experimentos de Northern blotting. A análise do transcriptoma de L. braziliensis revelou uma diferença de expressão gênica entre os estágios de desenvolvimento que variou entre 52% e 71%, dependendo dos estágios comparados. Também foram definidos os limites das 5UTRs e 3UTRs de 81% e 38% das CDSs anotadas, respectivamente. Propusemos uma metodologia para identificação de ncRNAs putativos utilizando dados de sequenciamento de RNA-total. Essa metodologia identificou 11.376 ncRNAs putativos em L. braziliensis, sendo que todos os candidatos foram analisados por programas preditores de características específicas de ncRNAs e apresentaram pelo menos uma predição positiva, além de não possuírem semelhança com domínios proteicos conhecidos. A análise de conservação demonstrou que de 27% a 41% dos ncRNAs putativos identificados são conservados em outras espécies de Leishmania. Foram encontrados de 27% a 38% de ncRNAs putativos com regulação atrelada ao estágio de desenvolvimento, dependendo dos estágios comparados. Assim, além da identificação e descrição de ncRNAs em Leishmania foram encontrados candidatos com regulação atrelada ao desenvolvimento e padrões foram descortinados com o processo de análise proposto e executado. Portanto, esse trabalho contribui significativamente para ampliar a compreensão dos processos de regulação de expressão gênica em Leishmania e oferecerá à comunidade um conjunto grande e importante de informações sobre a organização genética do parasito, diferenças genéticas e regulatórias ao longo do desenvolvimento, além de informações do transcriptoma de forma global. / High-throughput data analyses indicated that transcriptomes are more extensive and complex than previously supposed. Currently, it is evident that most eukaryotic genomes are almost entirely transcribed and under regulation tied to developmental stages. Control of gene expression involves regulatory non-coding RNAs (ncRNAs), including post-transcriptional processes. Post-transcriptional regulation is particularly relevant for the regulation of gene expression in trypanosomatids, as compared to other organisms. Parasites of the genus Leishmania (Order Kinetoplastidae, family Trypanosomatidae) causes infectious-parasitic diseases known as leishmaniasis, and their life cycle comprises three development stages: procyclic promastigotes, metacyclic promastigotes and amastigotes. This study aimed to computationally identify and characterize Leishmania putative ncRNAs at different stages of development. Large-scale approaches combined with bioinformatics tools allowed the identification of 11,376 putative ncRNAs in L. braziliensis and, in a preliminary study, of 37 putative ncRNAs in L. donovani. In addition, the L. braziliensis the complete transcriptome was analyzed comparatively between the parasite development stages. In L. donovani, of the 37 putative ncRNAs identified, 34 were in UTRs (Untranslated Regions) and 3 in intergenic regions. Predictors of ncRNAs specific characteristics were used and 32 putative ncRNAs had at least one positive prediction. All candidates are conserved, partially or entirely, in at least three Leishmania species. Five L. donovani ncRNAs were confirmed by Northern blotting experiments. Analysis of the L. braziliensis transcriptome revealed differences in gene expression levels between developmental stages, ranging from 52% to 71%, depending on the compared stages. The boundaries of the 5\'UTRs and 3\'UTRs were also defined for 81% and 38% of the annotated CDSs, respectively. We developed a methodology for the identification of putative ncRNAs using total RNA sequencing data. This methodology allowed the identification of 11,376 putative ncRNAs in L. braziliensis, and all candidates were analyzed by predictive programs of ncRNAs specific characteristics and presented at least one positive prediction, in addition to bearing no similarity to known protein domains. The analysis showed that from 27% to 41% of the putative ncRNAs identified are conserved in other Leishmania species. We found 27% to 38% of putative ncRNAs with regulation associated to the developmental stage, depending on the compared stages. Consequently, besides identification and characterization of ncRNAs in Leishmania, candidates with developmental-related regulation were found and patterns were uncovered with the proposed and implemented analysis. Thus, this work contributes significantly to improve understanding of gene expression regulation processes in Leishmania and will offer to the community important information about the parasite genetics and regulatory differences along the development, besides of L. braziliensis transcriptome information.

L’ARN non-codant 7SK forme la charpente d’un complexe, 7SK snRNP, qui régule l’activité du facteur d’élongation de la transcription P-TEFb, intervenant dans la levée des pauses transcriptionelles chez les métazoaires. Le 7SK snRNP comprend les protéines LARP7, essentielle pour la stabilité de l’ARN 7SK et MePCE, participant à sa coiffe. Dans le cadre d’une investigation du rapport entre structure et fonction de l’ARN7SK, le projet était de comprendre commen la protéine LARP7 reconnait et assemble l’ARN dans le 7SK snRNP. La protéine LARP7, membre d’une famille reliée à laprotéine La, est spécifique de 7SK. Les éléments responsables de l’interaction ont été analysés par des méthodes biochimiques dans des complexes reconstitués à partir d’ARN synthétique et de protéines recombinantes. Le module La, dans la région N-terminale, reconnaît et lie les trois uridines à l’extrémité 3’ de l’ARN et, additionellement,une séquence conservée au pied de la tigeboucle en 3’, induisant une conformation fermée de l’ARN. L’autre extrémité de la protéine comprend un domain RRM de reconnaissance de l’ARN, qui se lie à la boucle apicale de la tige-boucle 3’. La protéine LARP7 reconnaît également une région conservée au centre de l’ARN. Dans l’ensemble, LARP7 utiliserait ses domaines terminaux et central pour envelopper l’ARN et le stabiliser. Au cours de ces travaux, une interaction directe du domaine C-terminal avec la tige-boucle 5’ a également été mise en évidence. Celle-ci comprend le site de liaison à la HEXIM, la protéine qui déclenche l’interaction avec P-TEFb et un rôle fonctionnel de LARP7 est envisagé. / The non-coding RNA 7SK is the scaffold for the 7SK snRNP complex that regulates PTEFb, the positive transcription elongation factor, which relieves transcription pauses in metazoans. The 7SK snRNP comprises the proteins LARP7, essential for 7SK stability and MePCE, involved in capping. In the frame of an investigation of how the structure of the7SK RNA sustains its function, the project was to understand how is the RNA recognized and assembled in the 7SK snRNPby the associated protein LARP7. LARP7, a La-related protein is specific for 7SK. The elements responsible for the interaction were investigated by biochemical approaches in vitro with complexes reconstituted from purified recombinant proteins and transcribed RNA. The La-module of LARP7 recognizes and binds the triplet of uridines at the 3’-end of the 7SK RNA and additionally binds to a conserved region at the foot of the 3’-hairpin.This may stabilize a closed conformation of the 7SK. On the other end of the LARP7molecule, the C-terminal domain comprising a RRM (RNA Recognition Motif) binds to the apical loop of the 3’hairpin. Further investigations showed that a conserved region in the core of the RNA is also involved. On the whole, this strongly suggests thatLARP7 wraps around 7SK using its N terminal, C-terminal and linker domains to ensure the RNA stabilization into a functional core. In the course of the investigation, was revealed a direct interaction of the C-terminal domain of LARP7 with the 5’-hairpin of the RNA, which is responsible for 7SK function as it contains the binding site of HEXIM, the protein which bridges 7SK and P-TEFb. A possible functional role of LARP7 is envisioned.

The Pentatricopeptide Repeat (PPR) gene family produces RNA-binding proteins that target organellar transcripts. The PPR family is expanded in land plants, with nearly 450 genes identified in Arabidopsis thaliana. In plants with a Cytoplasmic Male Sterility (CMS) phenotype, members of the PPR family can act as a RESTORER OF FERTILITY (Rf) and are part of a subset of genes called RESTORER OF FERTILITY-LIKE (RFL). Unlike other PPR transcripts, RFL transcripts are targets of both microRNA (miRNA) and trans-acting siRNA (tasiRNA) and produce secondary siRNA after initial miRNA- or tasiRNA-guided cleavage. We utilized the A. lyrata genome assembly and high-throughput sequencing of small RNA to examine the evolutionary dynamics of the PPR gene family and the pattern of small RNA targeting of RFL transcripts. We found an expanded set of 539 PPR genes in A. lyrata, 51 of which were in the RFL group, often in multiple collinear copies when compared to their A. thaliana orthologs. In-species RFL paralogs appear to be more related to one another than to their collinear orthologs, which is possible evidence of gene conversion or ectopic recombination. miRNA targeting of RFL transcripts is largely conserved with nearly two-thirds of all target sites maintained. TasiRNA targeting was less conserved with roughly one-third of comparable validated tasiRNA targets maintained in both species. However, when clusters of potential tasiRNA targets were considered, roughly two-thirds of target sites are conserved. Production of secondary siRNA from A. lyrata PPR transcripts is less well defined than in A. thaliana, with strong signals coming from phases that are not concordant with the miRNA- or tasiRNA-guided cleavage sites. / Graduation date: 2013

Immer mehr Berichte deuten darauf hin, dass nicht-kodierende RNAs an der Regulation des Immunsystems beteiligt sind. In dieser Arbeit wurden nicht-kodierende RNAs identifiziert, die durch zwei unterschiedliche immunologische Prozesse in zwei verschiedenen Zelltypen reguliert wurden. Zum einen wurde das Transkriptom von Multiplen Myelom-Zellen in Abhängigkeit von der Interleukin 6-Stimulation untersucht. Dabei wurden einige sehr lange, IL 6-regulierte macroRNAs identifiziert, die STAIRs (STAT3-induced RNAs). Bei den STAIRs handelt es sich wahrscheinlich um funktionelle, kontinuierliche, nicht-kodierende macroRNAs, die im Zellkern angereichert sind. Einige STAIRs dienen eventuell zusätzlich oder ausschließlich als Primärtranskript für gespleißte, lange ncRNAs (lncRNAs), die weitere Funktionen in der Zelle ausüben können. Die STAIRs weisen eine große Bandbreite an Gewebsspezifität auf und bei den Untersuchungen in dieser Arbeit zeigten sich Hinweise, dass sie sich für verschiedene Krebserkrankungen als Biomarker eignen könnten. Die zweite Transkriptomanalyse wurde bei der Aktivierung naiver T Zellen durchgeführt. Dabei offenbarte sich, dass die Zellen bei diesem Prozess einen dramatischen Wechsel ihres Transkriptionsprogrammes vollziehen und eine Vielzahl nicht Protein-kodierender Gene reguliert werden. Es wurde die Regulation von ncRNAs, die bisher noch nicht im Zusammenhang mit T Zellen beschrieben wurden, beobachtet und erneut unbekannte, differentiell exprimierte Bereiche identifiziert. Im Anschluss wurde STAIR18, eine nicht-kodierende RNA, die durch die beiden untersuchten Signalwege reguliert wird, eingehender untersucht. Es zeigte sich, dass STAIR18 im menschlichen Genom dupliziert ist und beide Loci die gespleißte, lange ncRNA152 in diversen Varianten transkribieren. ncRNA152 ist hauptsächlich im Zytoplasma lokalisiert und befindet sich dort anscheinend in perinukleären Aggregaten. Die verschiedenen ncRNA152-Isoformen scheinen unter-schiedliche Funktionen auszuführen. Einerseits ist eine Wirkung als competing endogenous RNA wahrscheinlich. Eine weitere Aufgabe der ncRNA152 scheint darin zu bestehen, das STAT3-Primärtranskript zu stabilisieren oder dessen Prozessierung zu fördern.

The studies in this thesis describe the application of genotyping and allele specific expression analysis to genetic studies. The role of the gene NPC1 in Triglyceride metabolism was explored in mouse models and in humans on the population level in study I. NPC1 was found to affect hepatic triglyceride metabolism, and to be relevant for controlling serum triglyceride levels in mice and potentially in humans. In study II the utility of the HapMap CEU samples was investigated for tagSNP selection in six European populations. The HapMap CEU was found to be representative for tagSNP selection in all populations while allele frequencies differed significantly in the sample from Kuusamo, Finland. In study III the power of Allele specific expression as a tool for the mapping of cis-regulatory variation was compared to standard eQTL analysis, ASE was found to be the more powerful type of analysis for a similar sample size. Finally ASE mapping was applied to regions reported to harbour long non-coding RNAs and associated SNPs were compared to published trait-associations. This revealed strong cis-regulatory SNPs of long non-coding RNAs with reported trait or disease associations.

Proteins and RNAs are two of the most versatile macromolecules that carry out almost all functions within living organisms. In this thesis I have explored evolutionary and regulatory aspects of proteins and RNAs by studying their structures, modifications and interactions. In the first chapter of my thesis I investigate domain atrophy, a term I coined to describe large-scale deletions of core structural elements within protein domains. By looking into truncated domain boundaries across several domain families using Pfam, I was able to identify rare cases of domains that showed atrophy. Given that even point mutations can be deleterious, it is surprising that proteins can tolerate such large-scale deletions. Some of the structures of atrophied domains show novel protein-protein interaction interfaces that appear to compensate and stabilise their folds. Protein-protein interactions are largely influenced by the surface and charge complementarity, while RNA-RNA interactions are governed by base-pair complementarity; both interaction types are inherently different and these differences might be observed in their interaction networks. Based on this hypothesis I have explored the protein-protein, RNA-protein and the RNA-RNA interaction networks of yeast in the second chapter. By analysing the three networks I found no major differences in their network properties, which indicates an underlying uniformity in their interactomes despite their individual differences. In the third chapter I focus on RNA-protein interactions by investigating post-translational modifications (PTMs) in RNA-binding proteins (RBPs). By comparing occurrences of PTMs, I observe that RBPs significantly undergo more PTMs than non-RBPs. I also found that within RBPs, PTMs are more frequently targeted at regions that directly interact with RNA compared to regions that do not. Moreover disorderedness and amino acid composition were not observed to significantly influence the differential PTMs observed between RBPs and nonRBPs. The results point to a direct regulatory role of PTMs in RNA-protein interactions of RBPs. In the last chapter, I explore regulatory RNA-RNA interactions. Using differential expression data of mRNAs and lncRNAs from mouse models of hereditary hemochromatosis, I investigated competing regulatory interactions between mRNA, lncRNA and miRNA. A mutual interaction network was created from the predicted miRNA interaction sites on mRNAs and lncRNAs to identify regulatory RNAs in the disease. I also observed interesting relations between the sense-antisense mRNA-lncRNA pairs that indicate mutual regulation of expression levels through a yet unknown mechanism.

Staphylococcus aureus est une bactérie pathogène portée par 30% de la population humaine. Cette bactérie agressive est responsable d'1/5ème des maladies acquises à l’hôpital (infections nosocomiales). Le passage d’un état commensal (portage) à un état infectieux implique le contrôle de l’expression de facteurs de virulence (toxines, adhésines…) ; ce qui nécessite un large arsenal de régulateurs bactériens comprenant des protéines (facteurs de transcription) et des ARN régulateurs (ARNrég). Parmi ces derniers, l’ARN Srn_3610_SprC est impliqué, entre autres, dans la prévention de la phagocytose et dans l’atténuation de la virulence de la bactérie. Or, cet ARN, dont l’expression est habituellement faible, se retrouve fortement exprimé durant les premières minutes de la phagocytose. Le but de cette thèse a été d’identifier les régulateurs transcriptionnels de srn_3610_sprC. SarA, un des facteurs de transcription majeur de S. aureus impliqué dans de nombreuses étapes clé de la virulence (antibiorésistance, formation de biofilm…), a été caractérisé comme le répresseur fort de l’expression de srn_3610_sprC. L’identification du site de fixation de SarA sur le promoteur de ce gène a permis de révéler un second ARNrég, Srn_9340, dont l’expression est également réprimée par SarA. Dans les 2 cas, SarA empêche la fixation de l’ARN polymérase sur leur promoteur, entrainant un faible niveau de transcription. La recherche du signal permettant l’induction de la transcription de ces gènes via le décrochage de SarA est en cours. En parallèle, les données de fixation de SarA sur ces 2 promoteurs ont permis d’identifier de nouvelles cibles de SarA. Nous poursuivrons cette recherche de cibles via une analyse à haut débit par RNASeq. / Staphylococcus aureus is a bacterial pathogen responsible for about 1/5 of health-care associated infections. Nevertheless, 30% of humans are healthy carriers of this bacterium. Switch from commensal to infectious mode requires that virulence factors (toxins, adhesins), involved in S. aureus pathogenicity, are regulated by transcription factors (TF) and small non-coding RNA (sRNA). One of these sRNA, Srn_3610_SprC, has a key-role in prevention of phagocytosis and in attenuation of S. aureus virulence. Whereas srn_3610_sprC is usually poorly expressed, its expression is up-regulated during the first minutes of phagocytosis process. The aim of this thesis was to identify TF regulating srn_3610_sprC expression. We characterized SarA, the main TF of S. aureus, as a repressor of srn_3610_sprC transcription. Following SarA binding site determination, we highlighted a second sRNA (Srn_9340) also transcriptionally repressed by SarA. For both sRNA, SarA prevents RNA polymerase binding on their promoters. The next challenge will be to determine SarA derepression signal allowing high level of sRNAs transcription. Meanwhile, researches on SarA binding sequences allowed us to identify new SarA targets. To better understand SarA functions in S. aureus (antibiotic resistance, biofilm formation), we are now initiating a global study for the determination of SarA targets.

Au cours de ces 10 dernières années une nouvelle classe de petits ARN non-codants nommés "tRNA-derived fragments" (tRFs) a été caractérisée. Tandis que le rôle canonique des tRNA est bien connu, les raisons pour lesquels des fragments de tRNA s'accumulent dans la cellule restent inconnues. Actuellement, peu d'informations sont disponibles sur leurs biogenèses et leurs rôles biologiques, mais les preuves montrant leur importance dans la régulation de l'expression des gènes augmente régulièrement. Cependant, peu de données sont disponibles chez les plantes. A l'aide d’expérience de "deep-sequencing" et de northern blot nous avons confirmé l'existence d'une grande population en tRFs d'origine variée. A la suite de ces observations, trois questions sont établies. Tout d'abord, quelles sont les enzymes responsables de la biogenèse des tRFs. Ensuite, où les tRFs sont générés. Enfin, est-ce que les tRFs sont des sous-produits de la dégradation des tRNA ou ont-ils une fonction biologique? / During the last decade, a new class of small non-coding RNAs called tRNA-derived fragments (tRFs) has emerged. Whilst the canonic role of tRNA is well-known, the reason(s) why stable tRFs remains in the cell is unknown. Indeed, the number of tRFs has rapidly increased in various evolutionary divergent organisms. To date, only few data on their biogenesis and on their biological roles is known but their importance in the regulation of gene expression and in cell life is expanding. In plants, the existence of tRFs has also been reported but only few data are available. Using deep-sequencing on various small RNA libraries from Arabidopsis thaliana and Northern blots experiments, we confirmed the existence of a large but specific population of tRFs. Following these observations, three questions are addressed. First, what are the enzymes responsible for tRFs biogenesis, second where are tRFs generated and third, are tRFs merely degredation by-products or do they have biological functions?