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Recent studies have established important roles of de novo mutations (DNMs) in autism spectrum disorders (ASDs). Here, we analyze DNMs in 262 ASD probands of Japanese origin and confirm the "de novo paradigm" of ASDs across ethnicities. Based on this consistency, we combine the lists of damaging DNMs in our and published ASD cohorts (total number of trios, 4,244) and perform integrative bioinformatics analyses. Besides replicating the findings of previous studies, our analyses highlight ATP-binding genes and fetal cerebellar/striatal circuits. Analysis of individual genes identified 61 genes enriched for damaging DNMs, including ten genes for which our dataset now contributes to statistical significance. Screening of compounds altering the expression of genes hit by damaging DNMs reveals a global downregulating effect of valproic acid, a known risk factor for ASDs, whereas cardiac glycosides upregulate these genes. Collectively, our integrative approach provides deeper biological and potential medical insights into ASDs.

Loaded into ARGONAUTE(AGO) proteins, eukaryotic micro(mi)RNAs regulate gene expression via cleavage, translational repression, and/or accelerated decay of sequence‐complementary target transcripts. Despite their importance in development, cell identity maintenance and stress responses, how individual miRNAs contribute to spatial gene regulation within the complex cell mosaics formed in tissues/organs has remained inaccessible in any organism to date. We have developed a non‐invasive methodology to examine, at single‐cell‐type resolution, the AGO‐loading and activity patterns of entire miRNA cohorts in intact organs, applied here to the Arabidopsis root tip. A dual miRNAome–targetome analytical interface allowing intuitive data integration/visualization was developed as the basis for in‐depth investigations via single‐cell‐type experimentation. These uncovered an array of so far speculative or hitherto unknown types of spatial miRNA‐mediated gene regulation schemes, including via widespread cell‐to‐cell movement between contiguous layers of distinct identities. This study provides the proof of principle that minimally invasive, genome‐scale analysis of miRNA activities within and between single‐cell types of whole organs is achievable., CAB was supported by an EMBO long‐term fellowship (ALTF 728‐2009). Work was supported by a European Research Council Advanced Grant (Frontiers in RNAi‐II No. 323071) and a grant from the Swiss National Foundation (No. 310030B_152832) both attributed to O.V.

Dissertação de mestrado em Bioquímica, apresentado à Faculdade de Ciências a Universidade de Lisboa, 2019 Co-orientadora Luísa Romão, Departamento de Saúde Ambiental do Instituto Nacional de Saúde Doutor Ricardo Jorge Translation of mRNA into protein is one of the most important and costly processes of the cell, since it supports and regulates several cellular events. Under normal conditions, translation initiation takes place through the canonical mechanism, in which first occurs the recognition of the cap structure at the 5'end of the transcript, followed by the scan of the 5'UTR (untranslated region) until the identification of the initiation codon in a favourable context. However, during high energy cost cell events, such as mitosis, or during some stresses such as hypoxia, mechanisms are activated that decrease canonical translation. Thus, under conditions of inhibition of canonical translation, there are proteins whose synthesis is not affected and is maintained by alternative mechanisms. These include those in which ribosomal recruitment occurs without cap recognition and/or 5'UTR scanning. In these cases, ribosomal recruitment occurs through secondary structures present in mRNA 5'UTR, which interact with the 40S ribosomal subunit, and with or without the help of initiation factors, position it near to the initiation codon. Therefore, it was hypothesized that Argonaute proteins, more properly AGO1 (human Argonaute RNA-induced silencing complex catalytic component 1) could be translated by non-canonical mechanisms due to its importance in the biogenesis of the microRNAs and in the gene silencing. Previous results obtained in our laboratory using a bicistronic system, demonstrated that AGO1 5'UTR is able to mediate cap-independent translation of a reporter gene, even under stress conditions that inhibit canonical translation. Thus, the purpose of this work was to complete this research project, continuing to study the cap-independent translation mediated by the 5'UTR of AGO1. In this way, the first objective was to reinforce the previously obtained results. For this it was intended to show that the AGO1 5´UTR can mediate an alternative initiation mechanism in a bicistronic vector different from the one previously used. For this, the proposed bicistronic system contains two reporter genes, one corresponding to enhanced green fluorescent protein (EGFP) and another corresponding to a red fluorescent protein (mCherry). The characteristic of this system is that in the first cistron is the sequence of EGFP, which is translated by the canonical mechanism. The second cistron is the mCherry sequence that can only be translated in cap-independent manner. With this in mind, the strategy was to clone the sequence corresponding to AGO1 5´UTR, upstream of the mCherry and to verify if it was able to potentiate cap-independent translation of this fluorophore. In this thesis we present the cloning strategy developed to obtain this construct, although we were not able to complete this task. However, we present some possible solutions to complete these clonings. Other objective was to evaluate how the expression of Argonaute proteins, more properly the endogenous AGO1, varies during endoplasmic reticulum stress, which inhibits the canonical translation. For this purpose, we treated HCT116 cells (colorectal cancer-derived cell line) with thapsigargin, which is a drug that induces the stress of the endoplasmic reticulum and, consequently, the inhibition of the canonical translation. Our results demonstrated that treatment with the drug induces a decrease in the AGO1 and AGO2 mRNA levels, a decrease in the total protein amount of Argonautes (AGO1, AGO2, AGO3 and AGO4) and a decrease in the AGO1 protein. In a way, this is an inconclusive result for the study of non-canonical translation of this protein during stress conditions. However, we think that this result is mainly due to two factors. One has to do with the fact that the treatment with the drug decreases the AGO1 mRNA level, thus having less transcript available for translation, which may lead to less protein synthesis, albeit in an alternative way. The other factor is related to the possibility that the non-canonical translation mechanism is not as efficient in as the canonical way, which explains the observed decrease. Thus, in future experiments it will be important to test other stresses that inhibit canonical translation, but that do not induce changes in the amount of mRNA and test this experiment in other cell lines. Finally, we aim to predict what structural features make the AGO1 5´UTR succeed in promoting a non-canonical translation initiation mechanism and thus try to estimate the secondary structure adopted by this sequence. For this we submited AGO1 5'UTR sequence to an in silico analysis. Among other characteristics, we found that it has a percentage of guanines/cytosines of 72.3% and it is organized into a stable secondary structure, which has four stem loops. As a future perspective, we aim to experimentally validate this structural prediction through circular dichroism. Next was performed an in silico deletion analysis in which nucleotides were removed from the sequence in order to predict the structural dynamics and to check if there was any minimal sequence/structure required to maintain stable the spatial conformation of AGO1 5'UTR. By our analysis it was verified that when stem loop I or stem loop IV is removed, the structure does not seem to change much in relation to the original, indicating that the conformation remains stable when small deletions occur. To complement this in silico deletion analysis it is intended in the future to validate experimentally, in a bicistronic vector, the effect of this deletions. We believe that once these research lines have been completed, we will better understand the relevance of alternative translation mechanisms in the maintenance of synthesis of several proteins, including AGO1, during stress situations, and even during the tumorigenesis process. A expressão génica é um processo a partir do qual a informação genética contida no ácido desoxirribonucleico (DNA, do inglês deoxyribonucleic acid) é descodificada em proteínas que serão usadas pelos sistemas biológicos, para a sua sobrevivência, manutenção ou desenvolvimento. Esta é dividida em várias etapas, sendo que tudo se inicia no núcleo com a transcrição da informação contida no DNA em moléculas de ácido ribonucleico mensageiro (mRNA, do inglês messenger ribonucleic acid). À medida que vão sendo sintetizadas, as moléculas de mRNA sofrem um processamento, em que serão introduzidas modificações que serão importantes para estabilidade do transcrito e para as etapas a jusante. Entre estas modificações estão a introdução da estrutura cap m7G na extremidade 5´ do transcrito, a poliadenilaçao da extremidade 3´ e o splicing do mRNA. Uma vez processado, o mRNA maduro será transportado do núcleo para o citoplasma, onde será traduzido para proteína. Em condições normais, o mRNA de eucariotas é traduzido de uma forma canónica, em que inicialmente é necessário que exista o reconhecimento da estrutura cap na extremidade 5´ do transcrito. Desta forma, a tradução inicia-se com o recrutamento da subunidade ribossomal 40S e de fatores de iniciação (eIF, do inglês eukaryotic initiation factor) para a extremidade 5´ do mRNA, sendo que será este complexo, pela ação do eIF4E que reconhecerá o cap. Uma vez ligados, a subunidade ribossomal inicia o scan da região 5´ não codificante (UTR, do inglês untranslated region) até encontrar um codão de iniciação em contexto favorável. De seguida, dar-se-á a síntese da proteína que, terminada, a tradução será utilizada para o funcionamento celular. Contudo durante alguns processos energeticamente dispendiosos para a célula, como a mitose, ou em condições de stress, como a hipoxia, a tradução canónica é afetada. Isto ocorre porque muitas vezes estes estímulos provocam a inibição do reconhecimento da estrutura cap, comprometendo assim a iniciação da tradução. No entanto, existem mRNAs eucariotas cuja expressão não é comprometida aquando destes eventos, sendo que estes transcritos normalmente codificam para proteínas fundamentais para a célula ou de resposta a stress. Desta forma em condições de inibição da tradução canónica, existem proteínas cuja síntese não é afetada, sendo mantida por mecanismos alternativos. Entre estes estão incluídos aqueles em que ocorre o recrutamento ribossomal para o mRNA sem que haja o reconhecimento do cap e/ou scan da 5´UTR. Nestes casos, o recrutamento ribossomal ocorre através de estruturas secundárias presentes na 5´UTR do mRNA, que interagem com o ribossoma, e com o auxílio ou não de fatores proteicos, posicionam este junto do codão de iniciação. Em eucariotas já foram descritos vários mRNAs cuja tradução é mantida em condições de inibição da tradução canónica, sendo os mecanismos alternativos ativados por vários tipos de stress como a hipoxia, a limitação de nutrientes ou a apoptose. Assim, os mecanismos alternativos de iniciação da tradução têm um papel importante para a manutenção da homeostasia celular durante situações de stress. Em sentido contrário estes também podem ter um papel negativo, nomeadamente na promoção do processo de tumorigénese. Geralmente os ambientes tumorais apresentam hipoxia e limitação de nutrientes, que são condições que inibem a tradução canónica e promovem mecanismos alternativos de síntese proteica. Deste modo, as células cancerígenas têm uma vantagem, pois podem manter a síntese de vários oncogenes de uma maneira não-canónica. No nosso trabalho, colocou-se a hipótese de que as proteínas Argonaute, mais propriamente a AGO1 (do inglês human Argonaute RNA-induced silencing complex catalytic component 1) poderiam ser traduzidas por mecanismos não-canónicos em condições de inibição da iniciação da tradução dependente do reconhecimento do cap. Estas proteínas estão envolvidas na síntese dos miRNAs e no silenciamento génico, que por sua vez participam na resposta a alguns stresses e na manutenção do funcionamento celular. Assim, devido à sua importância propôs-se que a síntese da AGO1 possa ser mantida durante condições de stress celular por um mecanismo não-canónico. Resultados anteriores obtidos no nosso laboratório utilizando um vetor bicistrónico, demonstraram que a sequência correspondente à 5´UTR do mRNA da AGO1 é capaz de mediar a tradução independente da estrutura cap de um gene repórter. Também se verificou que esta atividade da 5´UTR da AGO1 é mantida tanto em condições normais, como em condições de stress celular que inibem a tradução canónica, como o stress do reticulo endoplasmático ou a hipoxia. Assim o propósito deste trabalho foi prosseguir nesta linha de investigação, continuando a estudar a tradução independente da estrutura cap mediada pela 5´UTR da AGO1. Desta forma, o primeiro objetivo foi reforçar os resultados obtidos anteriormente, pretendendo-se mostrar que a 5´UTR da AGO1 medeia um mecanismo de iniciação de tradução alternativo, num vetor bicistrónico diferente do utilizado anteriormente. Para isso o sistema bicistrónico utilizado contem dois genes repórteres, um correspondente à proteína fluorescente verde (EGFP, do inglês enhanced green fluorescent protein) e outro correspondente a uma proteína fluorescente vermelha (mCherry). A característica deste sistema é que no primeiro cistrão está a sequência da EGFP, que é traduzida de uma forma dependente da estrutura cap, ou seja, tradução canónica, enquanto que no segundo cistrão está a sequência do mCherry, que a ser traduzida, será duma forma independente da estrutura cap. Entre os dois cistrões está clonado um hairpin, de modo a prevenir a reiniciação da tradução. Tendo isto em mente, a estratégia foi clonar a sequencia correspondente à 5´UTR da AGO1 a montante do mCherry e verificar se esta conseguia potenciar a iniciação da tradução independente da estrutura cap. Nesta tese apresentamos a estratégia de clonagem desenvolvida para obter este constructo, ainda que sem sucesso aquando do término dos trabalhos. Contudo apresentamos algumas possíveis soluções para completar estas clonagens. Outro objetivo desta tese foi avaliar como varia a expressão das proteínas Argonaute, mais propriamente da AGO1 endógena, durante um stress que inibe a tradução canónica. A abordagem experimental foi tratar células HCT116 (linha celular derivada de cancro colorretal) com tapsigargina, que é uma droga que induz o stress do reticulo endoplasmático e, por consequência, a inibição da tradução canónica. Posteriormente foram analisados os efeitos do tratamento com esta droga nos níveis de mRNA das quatro Argonautes (AGO1, AGO2, AGO3 e AGO4), no nível de proteína do pool total de AGOs e no nível de proteína de AGO1. Os resultados obtidos demonstram que o tratamento com a tapsigargina induz uma diminuição nos níveis de mRNA da AGO1 e AGO2 para 54% e 62%, respetivamente. Um resultado que nós acreditamos ser uma consequência indireta da inibição da tradução induzida pela droga. Isto é, ao bloquear-se um processo tão determinante como a tradução, poderão ser afetados indiretamente outros processos celulares. Neste caso, parece indicar que a inibição da tradução canónica afeta a disponibilidade de dois transcritos, o da AGO1 e o da AGO2. Adicionalmente, verificou-se que o tratamento com esta droga afeta a quantidade total de proteínas Argonaute, uma vez que se observa a diminuição do pool de AGOs. Neste caso este resultado dever-se-á ao facto do tratamento com a tapsigargina induzir a diminuição dos níveis de mRNA da AGO1 e AGO2, fazendo com que haja menos transcrito disponível para a síntese destas proteínas. Com a expectável diminuição da quantidade de proteína AGO1 e AGO2, a consequência é a diminuição do pool de todas as AGOs. Finalmente, verificamos que o tratamento com a tapsigargina provoca a diminuição dos níveis proteicos de AGO1 endógena. De certa forma, este é um resultado inconclusivo para o estudo da tradução não-canónico desta proteína durante condições de stress. No entanto, pensamos que este resultado se deve principalmente a dois fatores. Um tem a ver com facto de o tratamento com a droga fazer diminuir o mRNA da AGO1, havendo por isso menos transcrito disponível para a tradução, o que poderá levar a uma menor síntese de proteína, mesmo que de uma forma alternativa. O outro fator está relacionado com a possibilidade de que o mecanismo de tradução não-canónico não ser tão eficiente como a via canónica, o que explica o decréscimo observado. Assim, em experiências futuras será importante testar outros tipos de stresses que inibam a tradução canónica, mas que não provoquem alterações na quantidade de mRNA e testar outras linhas celulares. Para além disso dever-se-á testar alguns stresses, que induzam maior atividade da 5´UTR da AGO1, em termos de síntese proteica. Por fim, o terceiro objetivo deste trabalho foi tentar prever quais as características estruturais que fazem com que a 5´UTR da AGO1 consiga promover um mecanismo não-canónico de iniciação da tradução. Desta forma tentar-se-ia aproximar a estrutura secundária adotada por esta região. Para isso fez-se uma análise in silico à sequência correspondente à 5´UTR da AGO1. Verificou-se que esta tem um comprimento de 213 nucleótidos, apresenta uma upstream ORF (do inglês open reading frame) e tem uma percentagem de guaninas/citosinas correspondente a 72.3%. Especialmente devido a este último fator a 5´UTR da AGO1 tem tendência a organizar-se numa estrutura secundária estável, que possui quatro stem loops. Como perspetiva futura, temos como objetivo validar experimentalmente esta previsão estrutural, através de dicroísmo circular. Posteriormente, fizemos uma análise delecional in silico, de modo a prever a dinâmica estrutural e verificar se havia alguma sequência/estrutura mínima necessária para manter a conformação espacial da 5´UTR da AGO1 estável. Para isso removeu-se, sequencialmente nucleótidos da 5´UTR da AGO1, tanto no sentido 5´ para 3´ como no sentido 3´ para 5´, e verificou-se qual o impacto que isso teria na estrutura da região comparativamente à estrutura com todo o comprimento. Pela nossa análise verificou-se que quando é removido o stem loop I ou o stem loop IV, a estrutura não parece alterar-se muito em relação à original, indicado que a conformação se mantém estável quando ocorrem pequenas deleções. Para complementar esta análise in silico, pretende-se no futuro validar experimentalmente estas previsões. Isto é verificar se a estrutura real adotada pela 5´UTR da AGO1 é semelhante à prevista computacionalmente, e analisar quais os efeitos de deleções na atividade desta sequência para mediar um mecanismo de tradução não-canónico, utilizando um vetor bicistrónico. Uma vez concluídas todas estas linhas de investigação pretender-se-á perceber de que forma é que o mecanismo de tradução alternativa de AGO1 pode estar envolvido no processo de tumorigénese. N/A

Tese de mestrado Bioquímica, Universidade de Lisboa, Faculdade de Ciências, 2019 Submitted by Cristina Manessiez (camanessiez@fc.ul.pt) on 2019-03-26T17:50:41Z No. of bitstreams: 1 ulfc124884_tm_Manuel_Carvalho.pdf: 2275283 bytes, checksum: c60e2e390f74d492d5850cda1762e1aa (MD5) Made available in DSpace on 2019-03-26T17:50:53Z (GMT). No. of bitstreams: 1 ulfc124884_tm_Manuel_Carvalho.pdf: 2275283 bytes, checksum: c60e2e390f74d492d5850cda1762e1aa (MD5) Previous issue date: 2019

Highlight The C-terminus of SiAGO1b is an essential motif for the interaction between SiAGO1b and SiHYL1, and plays a key role in regulating growth, development and stress responses in Setaria italic., Foxtail millet (Setaria italica (L.) P. Beauv), which belongs to the Panicoideae tribe of the Poaceae, is an important grain crop widely grown in Northern China and India. It is currently developing into a novel model species for functional genomics of the Panicoideae as a result of its fully available reference genome sequence, small diploid genome (2n=18, ~510Mb), short life cycle, small stature and prolific seed production. Argonaute 1 (AGO1), belonging to the argonaute (AGO) protein family, recruits small RNAs and regulates plant growth and development. Here, we characterized an AGO1 mutant (siago1b) in foxtail millet, which was induced by ethyl methanesulfonate treatment. The mutant exhibited pleiotropic developmental defects, including dwarfing stem, narrow and rolled leaves, smaller panicles and lower rates of seed setting. Map-based cloning analysis demonstrated that these phenotypic variations were attributed to a C–A transversion, and a 7-bp deletion in the C-terminus of the SiAGO1b gene in siago1b. Yeast two-hybrid assays and BiFC experiments revealed that the mutated region was an essential functional motif for the interaction between SiAGO1b and SiHYL1. Furthermore, 1598 differentially expressed genes were detected via RNA-seq-based comparison of SiAGO1b and wild-type plants, which revealed that SiAGO1b mutation influenced multiple biological processes, including energy metabolism, cell growth, programmed death and abiotic stress responses in foxtail millet. This study may provide a better understanding of the mechanisms by which SiAGO1b regulates the growth and development of crops.

To counteract host antiviral RNA silencing, plant viruses encode numerous viral suppressors of RNA silencing (VSRs). P0 proteins have been identified as VSRs in many poleroviruses. However, their suppressor function has not been fully characterized. Here, we investigated the function of P0 from pea mild chlorosis virus (PMCV) in the suppression of local and systemic RNA silencing via green fluorescent protein (GFP) co-infiltration assays in wild-type and GFP-transgenic Nicotiana benthamiana (line 16c). Amino acid deletion analysis showed that N-terminal residues Asn 2 and Val 3, but not the C-terminus residues from 230&ndash, 270 aa, were necessary for PMCV P0 (P0PM) VSR activity. P0PM acted as an F-box protein, and triple LPP mutation (62LPxx79P) at the F-box-like motif abolished its VSR activity. In addition, P0PM failed to interact with S-phase kinase-associated protein 1 (SKP1), which was consistent with previous findings of P0 from potato leafroll virus. These data further support the notion that VSR activity of P0 is independent of P0&ndash, SKP1 interaction. Furthermore, we examined the effect of P0PM on ARGONAUTE1 (AGO1) protein stability, and co-expression analysis showed that P0PM triggered AGO1 degradation. Taken together, our findings suggest that P0PM promotes degradation of AGO1 to suppress RNA silencing independent of SKP1 interaction.

RNA silencing is a conserved molecular mechanism in eukaryotes, of which the main effectors are the ARGONAUTE (AGO) proteins. In plants, AGO1 is a protein that is essential for growth and antiviral defence. It uses small RNAs as probe to recognize and regulate messenger RNAs. Viruses have developed suppressors of RNA silencing to overcome this defence. One of these, P0 from the Turnip Yellows Virus, acts as an F-box protein to hijack the SCF complex, an E3 ubiquitin ligase, and guide AGO1 to the ubiquitin-dependent proteolysis. This degradation uses the vacuole instead of the 26S proteasome, generally associated with ubiquitin-dependant proteolysis. This proteolysis mechanism is not understood and is also apparent when AGO1 is endogenously destabilized, suggesting that P0 uses an already existing pathway. An Arabidopsis F-box protein, FBW2, has been shown to impact AGO1 homeostasis independently from the proteasome. My PhD project aimed at characterizing FBW2 F-box activity and understanding the relationship between AGO1 and FBW2, as well as its consequences on the RNA silencing. The results obtained in this manuscript show that the SCFFBW2 interacts with AGO1 and triggers its degradation through an autophagy- and proteasome- independent process, while only weakly affecting the RNA silencing. FBW2 would actually target a subset of AGO1 proteins, which appears not to contain small RNAs. This regulation would play a surveillance role in order to prevent a deleterious activity of AGO1 in absence of small RNAs.; L'ARN interférence est un mécanisme moléculaire conservé chez les Eucaryotes dont les principaux acteurs sont les protéines ARGONAUTE (AGO). Chez les plantes, AGO1 est une protéine essentielle à la croissance et la défense antivirale. Elle utilise des petits ARNs comme sondes pour reconnaître et réguler des ARN messagers. Les virus ont développé des suppresseurs de l'ARN interférence pour surmonter cette défense. L'un d'entre eux, P0 du virus de la mosaïque jaune du navet, est comme une protéine F-box qui détourne le complexe SCF, une ubiquitine ligase E3, et conduit AGO1 vers la protéolyse ubiquitine-dépendante. Cette dégradation utilise la vacuole au lieu du protéasome 26S, généralement associé à la dégradation ubiquitine-dépendante. Ce mécanisme de protéolyse n'est pas compris et est aussi apparent quand AGO1 est déstabilisé de manière endogène, suggérant que P0 utilise une voie déjà existante. Une protéine F-box d'Arabidopsis, FBW2, a été décrite comme impactant l'homéostasie d'AGO1 indépendamment du protéasome. Mon projet de thèse visait à caractériser l'activité F-box de FBW2 et à comprendre la relation entre AGO1 et FBW2 ainsi que ses conséquences sur l'ARN interférence. Les résultats obtenus dans ce manuscrit montrent que le complexe SCFFBW2 interagit avec AGO1 et déclenche sa dégradation via un processus indépendant de l'autophagie ou du protéasome, tout en n'affectant que faiblement l'ARN interférence. FBW2 ciblerait en fait un sous-ensemble de protéines AGO1 qui semble ne pas contenir de petits ARNs. Cette régulation jouerait un rôle de surveillance pour prévenir une activité délétère d'AGO1 en absence de petits ARNs.

In all eukaryotes, regulated gene expression is key to orchestrate cell cycle progression. Not only genes encoding important core cell cycle regulators, but also genes of a variety of other factors involved in signal transduction, hormonal regulation and metabolic control are expressed at specific time points of the cell cycle. These changes in gene expression are controlled at multiple levels, including transcriptional and post-translational controls. In mammals, it became evident that microRNAs contribute to this regulation by targeting the transcripts of numerous cell cycle-regulated genes. However, in plants we still know little about the regulatory roles of small RNAs in the control of cell proliferation. During my thesis, I showed that depletion of Arabidopsis AGO1 impairs cell proliferation and root meristem activity. To further determine the repertoire and role of sRNAs in cell cycle regulation, we thus sequenced total RNAs and small RNAs, AGO1-associated small RNAs and the RNA degradome of synchronized BY2 cells at S-, G2-, M- and G1-phases of the cell cycle.; Chez tous les eucaryotes, la régulation de l’expression génique est primordiale pour le contrôle du cycle cellulaire. Un large éventail de gènes, incluant des régulateurs essentiels du cycle, mais aussi d’autre gènes impliqués dans la transduction du signal, la régulation hormonale et le métabolisme sont ainsi exprimé à certaines phases du cycle. Ces changements sont contrôlés à de multiples niveaux, notamment de façon transcriptionnelle et post-traductionnelle. Chez les mammifères, il est aujourd’hui évident que les micro ARNs contribuent à cette régulation en ciblant spécifiquement les transcrits d’un grand nombre de gènes régulés au cours du cycle. Cependant, nous n’avons que très peu d’informations à ce jour concernant le rôle des petits ARNs sur le contrôle de la prolifération cellulaire chez les plantes. Mes travaux de thèse ont permis de démontrer que la perte d’AGO1 affecte la prolifération cellulaire et l’activité du méristème racinaire. Nous avons également séquencé les transcrits, les petits ARNs et le dégradome à partir de cellules BY-2 synchronisées afin de déterminer le répertoire et la fonction des petit ARNs au cours du cycle cellulaire.

In all eukaryotes, regulated gene expression is key to orchestrate cell cycle progression. Not only genes encoding important core cell cycle regulators, but also genes of a variety of other factors involved in signal transduction, hormonal regulation and metabolic control are expressed at specific time points of the cell cycle. These changes in gene expression are controlled at multiple levels, including transcriptional and post-translational controls. In mammals, it became evident that microRNAs contribute to this regulation by targeting the transcripts of numerous cell cycle-regulated genes. However, in plants we still know little about the regulatory roles of small RNAs in the control of cell proliferation. During my thesis, I showed that depletion of Arabidopsis AGO1 impairs cell proliferation and root meristem activity. To further determine the repertoire and role of sRNAs in cell cycle regulation, we thus sequenced total RNAs and small RNAs, AGO1-associated small RNAs and the RNA degradome of synchronized BY2 cells at S-, G2-, M- and G1-phases of the cell cycle.; Chez tous les eucaryotes, la régulation de l’expression génique est primordiale pour le contrôle du cycle cellulaire. Un large éventail de gènes, incluant des régulateurs essentiels du cycle, mais aussi d’autre gènes impliqués dans la transduction du signal, la régulation hormonale et le métabolisme sont ainsi exprimé à certaines phases du cycle. Ces changements sont contrôlés à de multiples niveaux, notamment de façon transcriptionnelle et post-traductionnelle. Chez les mammifères, il est aujourd’hui évident que les micro ARNs contribuent à cette régulation en ciblant spécifiquement les transcrits d’un grand nombre de gènes régulés au cours du cycle. Cependant, nous n’avons que très peu d’informations à ce jour concernant le rôle des petits ARNs sur le contrôle de la prolifération cellulaire chez les plantes. Mes travaux de thèse ont permis de démontrer que la perte d’AGO1 affecte la prolifération cellulaire et l’activité du méristème racinaire. Nous avons également séquencé les transcrits, les petits ARNs et le dégradome à partir de cellules BY-2 synchronisées afin de déterminer le répertoire et la fonction des petit ARNs au cours du cycle cellulaire.

Tese de mestrado, Biologia Molecular e Genética, Universidade de Lisboa, Faculdade de Ciências, 2018 Submitted by Cristina Manessiez (camanessiez@fc.ul.pt) on 2019-02-05T19:06:07Z No. of bitstreams: 1 ulfc125212_tm_Diana_Barata.pdf: 2935909 bytes, checksum: 0c59e59c8c80b9712046386f51f2194d (MD5) Made available in DSpace on 2019-02-05T19:06:17Z (GMT). No. of bitstreams: 1 ulfc125212_tm_Diana_Barata.pdf: 2935909 bytes, checksum: 0c59e59c8c80b9712046386f51f2194d (MD5) Previous issue date: 2018

In plants, sugars such as glucose act as signalling molecules that promote changes in gene expression programmes that impact on growth and development. Recent evidence has revealed the potential importance of controlling mRNA decay in some aspects of glucose-mediated regulatory responses suggesting a role of microRNAs (miRNAs) in these responses. In order to get a better understanding of glucose-mediated development modulation involving miRNA-related regulatory pathways, early seedling development of mutants impaired in miRNA biogenesis (hyl1-2 and dcl1-11) and miRNA activity (ago1-25) was evaluated. All mutants exhibited a glucose hyposensitive phenotype from germination up to seedling establishment, indicating that miRNA regulatory pathways are involved in the glucose-mediated delay of early seedling development. The expression profile of 200 miRNA primary transcripts (pri-miRs) was evaluated by large-scale quantitative real-time PCR profiling, which revealed that 38 pri-miRs were regulated by glucose. For several of them, the corresponding mature miRNAs are known to participate directly or indirectly in plant development, and their accumulation was shown to be co-regulated with the pri-miR by glucose. Furthermore, the expression of several miRNA target genes was found to be deregulated in response to glucose in the miRNA machinery mutants ago1-25, dcl1-11, and hyl1-2. Also, in these mutants, glucose promoted misexpression of genes for the three abscisic acid signalling elements ABI3, ABI4, and ABI5. Thus, miRNA regulatory pathways play a role in the adjustments of growth and development triggered by glucose signalling.

International audience; RNA quality control (RQC) eliminates aberrant RNAs based on their atypical structure, whereas posttranscriptional gene silencing (PTGS) eliminates both aberrant and functional RNAs through the sequence-specific action of short interfering RNAs (siRNAs). The Arabidopsis thaliana mutant smd1b was identified in a genetic screen for PTGS deficiency, revealing the involvement of SmD1, a component of the Smith (Sm) complex, in PTGS. The smd1a and smd1b single mutants are viable, but the smd1a smd1b double mutant is embryo-lethal, indicating that SmD1 function is essential. SmD1b resides in nucleoli and nucleoplasmic speckles, colocalizing with the splicing-related factor SR34. Consistent with this, the smd1b mutant exhibits intron retention at certain endogenous mRNAs. SmD1 binds to RNAs transcribed from silenced transgenes but not nonsilenced ones, indicating a direct role in PTGS. Yet, mutations in the RQC factors UPFRAMESHIFT3, EXORIBONUCLEASE2 (XRN2), XRN3, and XRN4 restore PTGS in smd1b, indicating that SmD1 is not essential for but rather facilitates PTGS. Moreover, the smd1b mtr4 double mutant is embryo-lethal, suggesting that SmD1 is essential for mRNA TRANSPORT REGULATOR4-dependent RQC. These results indicate that SmD1 interplays with splicing, RQC, and PTGS. We propose that SmD1 facilitates PTGS by protecting transgene-derived aberrant RNAs from degradation by RQC in the nucleus, allowing sufficient amounts to enter cytoplasmic siRNA bodies to activate PTGS.

Eukaryotic Argonaute proteins play primary roles in miRNA and siRNA pathways that are essential for numerous developmental and biological processes. However, the functional roles of the four ZmAGO1 genes have not yet been characterized in maize (Zea mays L.). In the present study, ZmAGO1a was identified from four putative ZmAGO1 genes for further characterization. Complementation of the Arabidopsis ago1‐27 mutant with ZmAGO1a indicated that constitutive overexpression of ZmAGO1a could restore the smaller rosette, serrated leaves, later flowering and maturation, lower seed set, and darker green leaves at late stages of the mutant to the wild‐type phenotype. The expression profiles of ZmAGO1a under five different abiotic stresses indicated that ZmAGO1a shares expression patterns similar to those of Argonaute genes in rice, Arabidopsis, and wheat. Further, variation in ZmAGO1a alleles among diverse maize germplasm that resulted in several amino acid changes revealed genetic diversity at this locus. The present data suggest that ZmAGO1a might be an important AGO1 ortholog in maize. The results presented provide further insight into the function of ZmAGO1a.

The ARGONAUTE gene family is involved in the regulation of gene expression via the RNAi Silencing Complex (RISC). microRNA (miRNA) are 20–22bp RNAs that direct RISC to target genes. Several miRNA have been characterized in plants. Their roles include control of flowering time, floral organ identity, cell division patterns, and leaf polarity. ARGONAUTE1 (AGO1) is required for stem cell function and organ polarity, as is the closely related protein PINHEAD/ZWILLE (PNH/ZLL). Through phenotypic and double mutant analysis, we show that AGO1 regulates stem cell function via SHOOT MERISTEMLESS (STM). CUPSHAPED COTYLEDONS1 and 2 (CUC1 and CUC2) positively regulate STM and are targets of miRNA. The effect of AGO1 on leaf polarity is dependent, in part, on its role in meristem function revealed by interactions with ASYMMETRIC LEAVES1(AS1). AGO1 is required for full expression of LEAFY (LFY), APETALA1 (AP1) and AGAMOUS (AG). Flowering time is unaffected but floral meristem identity is partially restored in a curlyleaf (clf) background and this is not due to clf's affects on AG expression. CLF is over expressed in ago1, showing that the RNAi pathway regulates polycomb-type epigenetic modifiers.

Profiling small RNAs in soybean Williams 82 (rsv), susceptible to Soybean mosaic virus (SMV, the genus Potyvirus, family Potyviridae) strains G2 and G7, and soybean PI96983 (Rsv1), resistant to G2 but susceptible to G7, identified the microRNA miR168 that was highly overexpressed only in G7-infected PI96983 showing a lethal systemic hypersensitive response (LSHR). Overexpression of miR168 was in parallel with the high-level expression of AGO1 mRNA, high-level accumulation of miR168-mediated AGO1 mRNA cleavage products but with severely repressed AGO1 protein. In contrast, AGO1 mRNA, degradation products and protein remained without significant changes in G2- and G7-infected Williams 82. Moreover, knock-down of SGS3, an essential component in RNA silencing, suppressed AGO1 siRNA, partially recovered repressed AGO1 protein, and alleviated LSHR severity in G7-infected Rsv1 soybean. These results suggest that both miRNA and siRNA pathways are involved in G7 infection of Rsv1 soybean, and LSHR is associated with breakdown of AGO1 homeostasis.

BACKGROUND AND AIMS: The TERMINAL FLOWER 1 (TFL1) gene is pivotal in the control of inflorescence architecture in arabidopsis. Thus, tfl1 mutants flower early and have a very short inflorescence phase, while TFL1-overexpressing plants have extended vegetative and inflorescence phases, producing many coflorescences. TFL1 is expressed in the shoot meristems, never in the flowers. In the inflorescence apex, TFL1 keeps the floral genes LEAFY (LFY) and APETALA1 (AP1) restricted to the flower, while LFY and AP1 restrict TFL1 to the inflorescence meristem. In spite of the central role of TFL1 in inflorescence architecture, regulation of its expression is poorly understood. This study aims to expand the understanding of inflorescence development by identifying and studying novel TFL1 regulators. METHODS: Mutagenesis of an Arabidopsis thaliana line carrying a TFL1::GUS (beta-glucuronidase) reporter construct was used to isolate a mutant with altered TFL1 expression. The mutated gene was identified by positional cloning. Expression of TFL1 and TFL1::GUS was analysed by real-time PCR and histochemical GUS detection. Double-mutant analysis was used to assess the contribution of TFL1 to the inflorescence mutant phenotype. KEY RESULTS: A mutant with both an increased number of coflorescences and high and ectopic TFL1 expression was isolated. Cloning of the mutated gene showed that both phenotypes were caused by a mutation in the ARGONAUTE1 (AGO1) gene, which encodes a key component of the RNA silencing machinery. Analysis of another ago1 allele indicated that the proliferation of coflorescences and ectopic TFL1 expression phenotypes are not allele specific. The increased number of coflorescences is suppressed in ago1 tfl1 double mutants. CONCLUSIONS: The results identify AGO1 as a repressor of TFL1 expression. Moreover, they reveal a novel role for AGO1 in inflorescence development, controlling the production of coflorescences. AGO1 seems to play this role through regulating TFL1 expression.

[EN] The TERMINAL FLOWER 1 (TFL1) gene is pivotal in the control of inflorescence architecture in arabidopsis. Thus, tfl1 mutants flower early and have a very short inflorescence phase, while TFL1-overexpressing plants have extended vegetative and inflorescence phases, producing many coflorescences. TFL1 is expressed in the shoot meristems, never in the flowers. In the inflorescence apex, TFL1 keeps the floral genes LEAFY (LFY) and APETALA1 (AP1) restricted to the flower, while LFY and AP1 restrict TFL1 to the inflorescence meristem. In spite of the central role of TFL1 in inflorescence architecture, regulation of its expression is poorly understood. This study aims to expand the understanding of inflorescence development by identifying and studying novel TFL1 regulators. Mutagenesis of an Arabidopsis thaliana line carrying a TFL1::GUS (beta-glucuronidase) reporter construct was used to isolate a mutant with altered TFL1 expression. The mutated gene was identified by positional cloning. Expression of TFL1 and TFL1::GUS was analysed by real-time PCR and histochemical GUS detection. Double-mutant analysis was used to assess the contribution of TFL1 to the inflorescence mutant phenotype. A mutant with both an increased number of coflorescences and high and ectopic TFL1 expression was isolated. Cloning of the mutated gene showed that both phenotypes were caused by a mutation in the ARGONAUTE1 (AGO1) gene, which encodes a key component of the RNA silencing machinery. Analysis of another ago1 allele indicated that the proliferation of coflorescences and ectopic TFL1 expression phenotypes are not allele specific. The increased number of coflorescences is suppressed in ago1 tfl1 double mutants. The results identify AGO1 as a repressor of TFL1 expression. Moreover, they reveal a novel role for AGO1 in inflorescence development, controlling the production of coflorescences. AGO1 seems to play this role through regulating TFL1 expression., We thank Herve Vaucheret for the ago1-26 seeds, Antonio Serrano-Mislata for the pBTG6 construct, and Cristina Ferrandiz for critical reading of the manuscript. The collaboration of the IBMCP staff from the greenhouse, sequencing and microscopy facilities is also acknowledged. This work was supported by grants from the Spanish Ministerio de Ciencia e Innovacion (BIO2009-10876 and CSD2007-00057), the Spanish Ministerio de Economia y Competitividad (BFU2012-38929) and the Generalitat Valenciana (ACOMP2012-101). P.F.N. was supported by a fellowship from the I3P program of CSIC.