ARGONAUTE proteins (AGOs) are generally accepted as key components of the post transcriptional gene silencing mechanism, also involved in plant antiviral defense. Except for reports on the antiviral roles of AGO1, AGO2 and AGO7 in Arabidopsis, the exact roles played by the individual AGOs in other plant species are largely unknown. This research focused on the identification and characterization of AGOs involved in antiviral RNAi response to various viruses in N. benthamiana. Based on the temporal and spatial distribution of AGO transcripts in 3 and 8-week old plant root, stem and leaf tissues, expressions of NbAGO mRNAs were found to vary with age and tissue specificity. Plant endogenous AGO mRNAs were knocked down through virus induced gene silencing techniques using the Tobacco rattle virus vector system and posteriorly challenged with a GFP-chimeric virus construct deficient of a silencing suppressor. Unlike in control non-silenced plants, the Tomato bushy stunt virus construct deficient of its P19 silencing suppressor was consistently seen to exhibit a strong fluorescence on N. benthamiana plants silenced for NbAGOs 2 and X. Similar results were also obtained upon silencing of NbAGO2 using hairpin vector techniques. Comparable observations were also made when Tobacco mosaic virus GFP constructs were agroinfiltrated on NbAGO2 silenced plants further hinting the antiviral defense roles played by these AGOs. Agroinfiltration of Foxtailmosaic virus, Sunnhemp mosaic virus, and Turnip crinkle virus GFP chimeric constructs on NbAGO2 silenced N. benthamiana plants, however did not result in accumulation of GFP indicating the AGO antiviral defense specificity to TBSV and TMV. The results also hinted at a role for AGO7. Collectively my findings suggest that the expression of AGOs in N. benthamiana is tissue and age dependent, and that unlike in the model plant Arabidopsis where the main antiviral AGO is thought to be AtAGO1; in N. benthamiana, NbAGOs 2 and X seem to be involved in an antiviral defense role against TBSV and TMV with other AGOs perhaps contributing.

Epithelial ovarian cancer (EOC) is a leading cause of morbidity and mortality among women in the United States, and the etiology is incompletely understood. Common, low penetrant genetic variants such as single nucleotide polymorphisms (SNPs) likely contribute to a significant proportion of EOC. We examined whether SNPs in two understudied yet biologically important types of genes, mitochondrial-related and miRNA-related genes, may contribute to EOC susceptibility using data from a large, homogeneous study population of 1,815 EOC cases and 1,900 controls (frequency-matched on age-group and race/ethnicity) genotyped through stage 1 of an ongoing genome-wide association study. Inter-individual variation in genes involved in mitochondrial biogenesis was strongly associated with EOC risk (empirical P=0.050), especially for genes NRF1, PPARGC1A, MTERF, ESRRA, and CAMK2D. SNPs in several genes involved in the biogenesis of miRNAs (LIN28, LIN28B, AGO2, DICER, and DROSHA) also demonstrated associations with EOC risk; a joint meta-analysis and in vitro investigations reinforced evidence for a protective role of LIN28B rs12194974 (combined OR= 0.90, 95% CI: 0.82-0.98), a G>A SNP predicted to reside in a transcription factor binding site in the highly conserved LIN28B promoter. Our findings provide valuable insight into the pathogenesis of EOC, and support the consideration of variants in these genes as candidates when building risk prediction models. Most importantly, this work has provided a strong foundation for further lines of research that may aid in reducing the burden of this disease.

The genus Benyvirus includes the most important and widespread sugar beet viruses transmitted through the soil by the plasmodiophorid Polymyxa betae. In particular Beet necrotic yellow vein virus (BNYVV), the leading infectious agent that affects sugar beet, causes an abnormal rootlet proliferation known as rhizomania. Beet soil-borne mosaic virus (BSBMV) is widely distributed in the United States and, up to date has not been reported in others countries. My PhD project aims to investigate molecular interactions between BNYVV and BSBMV and the mechanisms involved in the pathogenesis of these viruses.BNYVV full-length infectious cDNA clones were available as well as full-length cDNA clones of BSBMV RNA-1, -2, -3 and -4. Handling of these cDNA clones in order to produce in vitro infectious transcripts need sensitive and expensive steps, so Ideveloped agroclones of BNYVV and BSBMV RNAs, as well as viral replicons allowing the expression of different proteins.Chenopodium quinoa and Nicotiana benthamiana plants have been infected with in vitro transcripts and agroclones to investigate the interaction between BNYVV and BSBMV RNA-1 and -2 and the behavior of artificial viral chimeras. Simultaneously I characterized BSBMV p14 and demonstrated that it is a suppressor of posttranscriptional gene silencing sharing common features with BNYVV p14.

Most free-living bacteria will encounter different environments and it is therefore critical to be able to rapidly adjust to new growth conditions in order to be competitively successful. Responding to changes requires efficient gene regulation in terms of transcription, RNA stability, translation and post-translational modifications. Studies of an extremely slow-growing mutant of Salmonella enterica, with a Glu125Arg mutant version of EF-Tu, revealed it to be trapped in a stringent response. The perceived starvation was demonstrated to be the result of increased mRNA cleavage of aminoacyl-tRNA synthetase genes leading to lower prolyl-tRNA levels. The mutant EF-Tu caused an uncoupling of transcription and translation, leading to increased turnover of mRNA, which trapped the mutant in a futile stringent response. To examine the essentiality of RNase E, we selected and mapped three classes of extragenic suppressors of a ts RNase E phenotype. The ts RNase E mutants were defective in the degradation of mRNA and in the processing of tRNA and rRNA. Only the degradation of mRNA was suppressed by the compensatory mutations. We therefore suggest that degradation of at least a subset of cellular mRNAs is an essential function of RNase E. Bioinformatically, we discovered that the mRNA of tufB, one of the two genes encoding EF-Tu, could form a stable structure masking the ribosomal binding site. This, together with previous studies that suggested that the level of EF-Tu protein could affect the expression of tufB, led us to propose three models for how this could occur. The stability of the tufB RNA structure could be affected by the elongation rate of tufB-translating ribosomes, possibly influenced by the presence of rare codons early in the in tufB mRNA. Using proteomic and genetic assays we concluded that two previously isolated RNAP mutants, each with a growth advantage when present as subpopulations on aging wild-type colonies, were dependent on the utilization of acetate for this phenotype. Increased growth of a subpopulation of wild-type cells on a colony unable to re-assimilate acetate demonstrated that in aging colonies, acetate is available in levels sufficient to sustain the growth of at least a small subpopulation of bacteria.

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.

In diverse eukaryotic organisms, double-stranded RNA (dsRNA) induces robust silencing of cellular RNA cognate to either strand of the input dsRNA; a phenomenon now known as RNA interference (RNAi). Within the RNAi pathway, small, 21 nucleotide (nt) duplexed RNA, dubbed small interfering RNAs (siRNAs), derived from the longer input dsRNA, guide the RNA induced silencing complex (RISC) to destroy its target RNA. Due to its ability to silence virtually any gene, whether endogenous or exogenous, in a variety of model organisms and systems, RNAi has become a valuable laboratory tool, and is even being heralded as a potential therapy for an array of human diseases. In order to understand this complex and unique pathway, we have undertaken the biochemical characterization of RNAi in the model insect, Drosophila melanogaster. To begin, we investigated the role of ATP in the RNAi pathway. Our data reveal several ATP-dependent steps and suggest that the RNAi reaction comprises as least five sequential stages: ATP-dependent processing of double-stranded RNA into siRNAs, ATP-independent incorporation of siRNAs into an inactive ~360 kDa protein/RNA complex, ATP-dependent unwinding of the siRNA duplex to generate an active complex, ATP-dependent activation of RISC following siRNA unwinding, and ATP-independent recognition and cleavage of the RNA target. In addition, ATP is used to maintain 5´ phosphates on siRNAs, and only siRNAs with these characteristic 5´ phosphates gain entry into the RNAi pathway. Next, we determined that RISC programmed exogenously with an siRNA, like that programmed endogenously with microRNAs (miRNAs), is an enzyme. However, while RISC behaves like a classical Michaelis-Menten enzyme in the presence of ATP, without ATP, multiple rounds of catalysis are limited by release of RISC-produced cleavage products. Kinetic analysis of RISC suggests that different regions of the siRNA play distinct roles in the cycle of target recognition, cleavage and product release. Bases near the siRNA 5´ end disproportionately contribute to target RNA-binding energy, whereas base pairs formed by the central and 3´ region of the siRNA provide helical geometry required for catalysis. Lastly, the position of the scissile phosphate is determined during RISC assembly, before the siRNA encounters its RNA target. In the course of performing the kinetic assessment of RISC, we observed that when siRNAs are designed with regard to 'functional asymmetry' (by unpairing the 5´ terminal nucleotide of the siRNA's guide strand, i.e. the strand anti-sense to the target RNA), not all of the RISC formed was active for target cleavage. We observed, somewhat paradoxically, that increased siRNA unwinding and subsequent accumulation of single-stranded RNA into RISC led to reduced levels of active RISC formation. This inactive RISC did not act as a competitor for the active fraction. In order to characterize this non-cleaving complex, we performed a series of protein-siRNA photo-crosslinking assays. From these assays we found that thermodynamic stability and termini structure plays a role in determining which proteins an siRNA will associate with, and how association occurs. Furthermore, we have found, by means of the photo-crosslinking assays, that siRNAs commingle with components of the miRNA pathway, particularly Ago1, suggesting overlapping functions or crosstalk for factors thought to be involved in separate, distinct pathways.

Submitted by Karin Goebel (karing@fiocruz.br) on 2014-11-25T18:05:56Z No. of bitstreams: 1 Dissertação Bruna Hilzendeger Marcon.pdf: 6455497 bytes, checksum: 8ea632ce91cdf16edd8b86a624972dba (MD5) / Approved for entry into archive by Karin Goebel (karing@fiocruz.br) on 2014-11-25T18:06:29Z (GMT) No. of bitstreams: 1 Dissertação Bruna Hilzendeger Marcon.pdf: 6455497 bytes, checksum: 8ea632ce91cdf16edd8b86a624972dba (MD5) / Made available in DSpace on 2014-11-25T18:06:29Z (GMT). No. of bitstreams: 1 Dissertação Bruna Hilzendeger Marcon.pdf: 6455497 bytes, checksum: 8ea632ce91cdf16edd8b86a624972dba (MD5) Previous issue date: 2014 / Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil / As células-tronco (CTs) caracterizam-se por possuírem a capacidade de se autorrenovar e de dar origem a um ou mais tipos celulares diferenciados. Nos últimos anos, diversos trabalhos mostraram a existência de CTs em tecidos adultos, tornando-as uma alternativa interessante para uso em terapias celulares. Contudo, para melhor utilizar as CTs, é preciso primeiramente compreender como ocorre a diferenciação em um tipo celular específico e, principalmente, como é regulada a expressão gênica durante este processo. Em 2009, Ingolia e colaboradores apresentaram uma nova técnica conhecida como ribosome profiling, a qual consiste no isolamento e sequenciamento em larga escala dos fragmentos de RNA associados e protegidos pelos ribossomos, os quais têm um tamanho aproximado de 30 nucleotídeos (conhecido com footprint ribossomal). Ao mapear as sequências obtidas, é possível obter informações não apenas sobre quais sequências estão sendo traduzidas, mas também sobre a cinética da tradução e sua extensiva rede de regulação. Assim, o objetivo deste trabalho foi aplicar a técnica de ribosome profiling ao estudo do processo de diferenciação de CTs adultas. Como modelo de estudo, foram utilizadas CTs obtidas de tecido adiposo antes (t=0) e após a indução para diferenciação adipogênica por 3 dias (t=72h). O primeiro passo do trabalho foi a adaptação do protocolo de ribosome profiling para o estudo de CTs adultas, o qual consiste na lise celular, digestão do lisado com uma RNA nuclease (a qual irá degradar o RNA exposto, preservando os fragmentos protegidos pelo ribossomo), ultracentifrugação do homogenato sobre colchão de sacarose 1 M para sedimentação dos ribossomos, extração de RNA e isolamento dos fragmentos de 30 nucleotídeos. Também foi feita extração do RNA poliA. As amostras foram sequenciadas (SOLiD™) e os dados obtidos foram triados e mapeados contra um banco de dados de RNAm, utilizando-se a ferramenta CLC Genomics Workbench. Foram identificados mais de 8.000 transcritos para as amostras de ribosome profiling e mais de 17.000 para as de poliA. Ao calcular o fold change entre as condições t=0 e t=72h, foi possível verificar que mais de 50% dos genes foram detectados como diferencialmente expressos apenas por ribosome profiling. Observou-se que genes relacionados com vias de diferenciação adipogênica e de metabolismo de lipídeos encontravam-se regulados positivamente em ambas as amostras de RNA. Por outro lado, observou-se que vias de regulação do citoesqueleto de actina e de adesão focal estavam reguladas negativamente apenas nas amostras de ribosome profiling. Isso é interessante, uma vez que a inibição destas vias já foi descrita como importante para o processo de adipogênese. Além disso, foi observada uma forte redução na eficiência de tradução de genes relacionados com a tradução após 72 horas de indução para diferenciação. Os resultados obtidos no presente trabalho reforçam as evidências de que os mecanismos de regulação pós-transcricionais e traducionais têm um papel muito importante na regulação da diferenciação celular de CTs, sendo que a técnica de ribosome profiling permitiu obter informações mais detalhadas de como este processo pode estar acontecendo. / Stem cells (SC) are characterized by their capacity of both self-renewing and giving rise to new differentiated cells. SC are found in adult tissues, which are considered a putative source for cell therapy. However, little is known about the mechanisms involved in the trigger of SCs differentiation into a specific cell type. Understanding adult SCs differentiation process is a fundamental step to better use and to take advantage of their potential. In 2009, Ingolia and collaborators presented a new methodology of transcriptome analysis named ribosome profiling, which consists on the isolation and deep-sequencing of the mRNA fragments enclosed by ribosomes. When lysed cells are submitted to nuclease digestion, unprotected mRNA is degraded, while fragments within ribosomes are preserved and have a known footprint of 30 nucleotides. Sequencing these ribosome-protected fragments results in a high-precision measurement of in vivo translation, providing precise information about translation kinetics and its extensive regulation. The objective of this work was to apply the ribosome profiling methodology to the study of adipogenic differentiation in adult SCs. SCs were isolated from human adipose tissue from three donors and were cultured in a control medium (t=0) and induced to adipogenic differentiation for 72 hours (t=72h). The first step was to adapt and optimize the ribosome profiling protocol to the SC model, which consists in cell lysis, cell lysate digestion by nuclease (to degrade unprotected RNA, preserving ribosome-protected fragments), ultracentrifugation over a 1M sucrose cushion to pellet ribosomes, RNA extraction and 30 nucleotides fragments isolation. poliA RNA was also isolated. Samples were submitted to deep-sequencing (SOLiD™) and the reads obtained were trimmed and mapped onto the reference mRNA database using the CLC Genomics Workbench. Over 8000 transcripts were identified in ribosome profiling samples and over 17000 in poliA samples. Fold change analysis between t=0 and t=72h of both RNA samples showed that differential expression of more than 50% of the genes was identified only by ribosome profiling. Pathways related to adipogenesis and lipid metabolism were upregulated in both RNA samples. However, regulation of the actin cytoskeleton and focal adhesion proteins were downregulated only in ribosome profiling samples. Interestingly, downregulation of these pathways was already described as an important phenomenon to cell adipogenesis. Besides, we observed a strong reduction of translational efficiency of genes involved in translation at t=72h. Our results reinforce previous data, suggesting that posttranscriptional and translational regulation play a fundamental role in the regulation of SC differentiation process and that ribosome profiling is an important tool to better understand this process.

L’inhibition, par p16INK4a, de la progression du cycle cellulaire est considérée comme liée à un arrêt de la progression en phase G1 du à l’inhibition de l’activité de CDK4/6. Nous montrons que l’expression ectopique de p16INK4a dans trois lignées cellulaires malignes, p16-/- et pRb+/+, issues de tissus différents, provoque un allongement de la durée de la phase S et du cycle cellulaire total. L’ensemble de nos travaux sur p16INK4a sauvage et son mutant p16G101W indique que p16INK4a induit un allongement de la phase S i) indépendamment de l’origine tissulaire des cellules analysées et ii) en partie lié aux conséquences de l’inhibition de l’activité de CDK4/6 et peut-être des MAP-kinases. Dans sa localisation nucléaire, p16INK4a interviendrait dans la régulation du cycle cellulaire indépendamment de sa liaison à CDK4. L’expression de CDK1 est inhibée par p16INK4a dans les trois lignées analysées. Dans les cellules MCF7 et U87, cette inhibition est post-transcriptionnelle, médiée par la région 3’non traduite de l’ARNm de CDK1, et est associée à une modification de l’équilibre d’expression, tissu-spécifique, des microARN régulant potentiellement CDK1. Nous démontrons que CDK1 est une cible de miR- 410 et miR-650 induits par p16INK4a et le rôle de l’inhibition de la voie pRb/E2F par p16INK4a dans l’induction de miR-410. Ainsi, p16INK4a régule l’expression des gènes à différents niveaux en modifiant l’équilibre fonctionnel des facteurs de transcription et, en conséquence, des miARN. / The inhibition of cell cycle progression by p16INK4a, have been considered to result from arrest in G1 phase due to inhibition of CDK4/6 activity. We show that ectopic expression of p16INK4a in three human malignant cell lines, p16-/- and pRb +/ +, derived from different tissues, led to an increase in the length of S phase and of the entire cell cycle. Our studies using wild-type p16INK4a and p16G101W mutant indicated that p16INK4a induces a lengthening of S phase i) independently of tissue origins and ii) partly linked to the inhibition of CDK4/6 activity and possibly MAP-kinases. In the nucleus, p16INK4a may intervene in regulating the cell cycle independently of binding to CDK4. The expression of CDK1 is inhibited by p16INK4a in three cell lines analyzed. In MCF7 and U87cells, this inhibition is post-transcriptional, mediated by the 3' non translated region of CDK1 mRNA, and is associated with changes in the balance of the expression of microRNAs, which regulate potentially CDK1. We demonstrate that CDK1 is a target of miR-410 and miR-650, both induced by p16INK4a and the role of the inhibition of pRb/E2F pathway by p16INK4a in the induction of miR-410. Thus, p16INK4a regulates gene expression at different levels by modifying the functional balance of transcription factors and, consequently, the microRNAs

Orientador: Ney Lemke / Resumo: MicroRNAs (miRNAs) são pequenas moléculas de RNA que regulam pós-transcricionalmente a expressão de genes, modelando o transcriptoma e a produção de proteínas. Em geral, os miRNAs são conservados no genoma de eucariotos, sendo considerados elementos vitais em diversos processos biológicos durante o desenvolvimento, tais como crescimento, diferenciação e morte celular. A grande diversidade de miRNAs identificados está restrita a poucas espécies e apenas uma parte do total de alvos de miRNAs preditos foi caracterizada funcionalmente. Nesse contexto, o uso da tecnologia de sequenciamento de alto rendimento (high throughput sequencing) atrelada à análise de nível transcricional por RT-qPCR possibilitam a identificação do microRNoma. A tilápia do Nilo, Oreochromis niloticus, é considerada um excelente modelo biológico para o estudo de miRNAs em vertebrados devido à sua importância econômica e evolutiva. O presente trabalho teve como objetivos: organizar os dados do sequenciamento dos miRNAs da tilapia do Nilo; disponibilizá-los em forma de uma base de dados para a comunidade científica; integrar as informações dos miRNAs identificados com outros bancos de dados de miRNAs; analisar os dados através de análises de bioinformática para determinação de agrupamentos definidos pelo nível de expressão de cada miRNA em seis tipos de tecido (músculo branco, músculo vermelho, testículo, ovário, fígado, olho, cérebro e coração) com distinção entre os gêneros e nas fases do desenvolvimento (2,... (Resumo completo, clicar acesso eletrônico abaixo) / Doutor

Submitted by Cláudia Bueno (claudiamoura18@gmail.com) on 2015-11-12T18:06:58Z No. of bitstreams: 2 Dissertação - Iane de Oliveira Pires Porto - 2014.pdf: 1352493 bytes, checksum: 3e4c1213c96035cbae32d6eeccdd14e5 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2015-11-13T10:33:50Z (GMT) No. of bitstreams: 2 Dissertação - Iane de Oliveira Pires Porto - 2014.pdf: 1352493 bytes, checksum: 3e4c1213c96035cbae32d6eeccdd14e5 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-11-13T10:33:50Z (GMT). No. of bitstreams: 2 Dissertação - Iane de Oliveira Pires Porto - 2014.pdf: 1352493 bytes, checksum: 3e4c1213c96035cbae32d6eeccdd14e5 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2014-02-19 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / MicroRNAs (miRNAs) are small non-coding RNAs involved in post-transcriptional expression regulation by inducing mRNA degradation or translation inhibition. Some miRNAs are known to regulate HLA-G expression, an important immunemodulatory molecule that inhibits both Natural Killer and cytotoxic T cells through interaction with inhibitory receptors. The HLA-G is associated with maternal-fetal tolerance, tissue acceptance in transplants and the progression of tumors. The mechanisms underlying HLA-G expression control are not completely understood, however, its 3’untranslated region (3’UTR) is reported to play an important role on gene regulation influencing mRNA stability and interacting with miRNAs such as miR-148a-3p. In this study, we performed a systematic analysis of all miRNAs that are good candidates to act as HLA-G regulators. In order to determine the miRNAs with the highest potential to influence HLA-G expression, we compared the outputs of three distinct algorithms - miRanda, RNAhybrid and Pita. For this purpose, a method of miRNA inference was developed using Perl scripts to compare and filter results and a scoring system was created in order to evaluate both the binding stability of the miRNA/mRNA interaction and the miRNA specificity to its target sequence. Then, a panel of miRNAs with great potential of controlling HLA-G expression was generated. / MicroRNAs (miRNAs) são pequenos RNAs não codificantes envolvidos na regulação gênica pós-transcricional por meio da degradação da molécula de RNA mensageiro ou da inibição da tradução. Alguns miRNAs foram relatados como sendo responsáveis pela regulação da expressão do gene HLA-G, um importante imunomodulador que inibe a ação de células Natural Killer e células T citotóxicas ao interagir com receptores inibitórios. Este gene está associado à tolerância maternofetal, aceitação de tecidos após transplantes e progressão de tumores. Os mecanismos subjacentes à regulação da expressão de HLA-G não foram completamente elucidados, mas sabe-se que sua região 3’ não traduzida (3’NT) possui um papel importante na regulação gênica tanto por manter a estabilidade da molécula de mRNA quanto por interagir com miRNAs como miR-148a-3p. Neste estudo, foram inferidos miRNAs que são bons candidatos para atuarem como reguladores do gene HLA-G. Para determinar os miRNAs com o maior potencial de operarem no controle pós-transcricional dos níveis de HLA-G, comparamos os resultados de três algoritmos distintos – miRanda, RNAhybrid e Pita. Para tanto, foi desenvolvida uma estratégia de inferência de miRNAs que utiliza scripts em Perl para comparação e filtragem dos dados e um sistema de pontuação que permite avaliar tanto a estabilidade da interação miRNA/mRNA quanto a especificidade do miRNA à sua sequência alvo. Assim, um painel confiável de miRNAs com grande possibilidade de influenciar a expressão de HLA-G foi gerado considerando as regiões polimórficas e não polimórficas da região 3’NT do gene HLA-G individualmente.