Dicer é um membro da família RNase III que controla a maturação de microRNA. A up-regulação de Dicer está associada a características agressivas e proliferação do melanoma. Nosso objetivo foi avaliar a expressão de Dicer em amostras de pacientes com melanoma nos diferentes estadios e em linhagens celulares de melanoma correlacionando a expressão de Dicer com progressão tumoral e resistência à apoptose. Comparamos Dicer no tecido de pacientes diagnosticados com melanoma em diferentes estadios, os resultados sugerem que o aumento da expressão de Dicer está associado à progressão do melanoma. Avaliamos nas linhagens knockdown para dicer1 a proliferação, capacidade clonogênica e sobrevida global. A linhagem celular SK-MEL-5 DICER KD foi mais suscetível à apoptose após o tratamento com cisplatina. Essa sensibilidade pode ser pela regulação dos receptores de morte, FAS e TNFR1, que estão envolvidos na indução de apoptose. Concluímos que o aumento na expressão de Dicer está relacionado com a progressão do Melanoma e com a resistência ao tratamento com cisplatina. / Dicer is a member of the RNase III family that controls the maturation of microRNA. Up-regulation of Dicer is is only associated with melanoma. The aim of this study was to evaluate the expression of Dicer in samples of patients diagnosed with melanoma at different stages of the disease and in melanoma cell lines correlating the expression of Dicer with tumor progression and resistance to apoptosis. We compared Dicer protein in the tissue of patients diagnosed with melanoma at different stages. We observed that the increased dicer expression is associated with melanoma progression. Cell proliferation, clonogenic capacity and overall survival were evaluated in the knockdown cell lines for dicer1. The SK-MEL-5 DICER KD cell line was more susceptible to apoptosis after treatment with cisplatin. The regulation of FAS and TNFR1 death receptors expressed by knockdown cell line were important in the induction of apoptosis. We conclude that the increase in Dicer expression is related to the progression of Melanoma and resistance to treatment with cisplatin.

MicroRNAs (miRNAs) are non-coding small RNAs that regulate eukaryotic gene activity at the post-transcriptional level by a process termed miRNA gene suppression. MicroRNA-122 (miR-122) is predominantly expressed in human liver cells and recent studies indicated that miR-122 promotes Hepatitis C Virus (HCV) replication and translation through physical interaction with two tandem binding sites located in the 5 untranslated region (5UTR) of the HCV genome (Jopling, et al., 2006; Jopling, et al., 2008). It has been reported that host genes that are also implicated in the miRNA gene suppression pathway are key regulators of HCV replication (Randall, et al., 2007). Two proteins, Dicer, a key RNaseIII enzyme, and its binding partner TRBP are essential proteins for miRNA activity. They are part of a protein complex called the RNA induced silencing complex (RISC) which also includes Argonaute proteins, and function in miRNA biogenesis loading the miRNA into RISC. As such, they are intriguing targets to study host-viral interplay during HCV replication.<p> In our study, we designed siRNAs to knock down Dicer and TRBP and then observed the effects of gene knockdown on full length J6/JFH-1-RLuc HCV (genotype 2a chimeric genome) replication and translation. The results showed that knocking down Dicer and TRBP reduced wild type (wt) J6/JFH-1-RLuc replication but had almost no effects on HCV translation in human liver cells. However, since knocking down Dicer and TRBP did not significantly alter miR-122 levels in the cell, it appears that the role of Dicer and TRBP was not solely the biogenesis of miR-122. This was confirmed by an experiment in which we observed that knocking down Dicer and TRBP also attenuated replication of a mutant virus in which replication is dependent on a exogenously supplied miRNA instead of endogenous miR-122.<p> Taken together, the results supported the hypotheses that Dicer and TRBP facilitate HCV infection mainly through HCV replication but not translation. The effects of Dicer and TRBP on HCV replication are not solely due to miR-122 biogenesis, and may be due to RISC loading functions in steps of miRNA gene suppression.<p> This study has set some essential groundwork for investigating potential roles of host factors in the RNAi machinery modulating HCV replication/translation and exploring novel antiviral targets.

MicroRNAs (miRNAs) are non-coding small RNAs that regulate eukaryotic gene activity at the post-transcriptional level by a process termed miRNA gene suppression. MicroRNA-122 (miR-122) is predominantly expressed in human liver cells and recent studies indicated that miR-122 promotes Hepatitis C Virus (HCV) replication and translation through physical interaction with two tandem binding sites located in the 5 untranslated region (5UTR) of the HCV genome (Jopling, et al., 2006; Jopling, et al., 2008). It has been reported that host genes that are also implicated in the miRNA gene suppression pathway are key regulators of HCV replication (Randall, et al., 2007). Two proteins, Dicer, a key RNaseIII enzyme, and its binding partner TRBP are essential proteins for miRNA activity. They are part of a protein complex called the RNA induced silencing complex (RISC) which also includes Argonaute proteins, and function in miRNA biogenesis loading the miRNA into RISC. As such, they are intriguing targets to study host-viral interplay during HCV replication.<p> In our study, we designed siRNAs to knock down Dicer and TRBP and then observed the effects of gene knockdown on full length J6/JFH-1-RLuc HCV (genotype 2a chimeric genome) replication and translation. The results showed that knocking down Dicer and TRBP reduced wild type (wt) J6/JFH-1-RLuc replication but had almost no effects on HCV translation in human liver cells. However, since knocking down Dicer and TRBP did not significantly alter miR-122 levels in the cell, it appears that the role of Dicer and TRBP was not solely the biogenesis of miR-122. This was confirmed by an experiment in which we observed that knocking down Dicer and TRBP also attenuated replication of a mutant virus in which replication is dependent on a exogenously supplied miRNA instead of endogenous miR-122.<p> Taken together, the results supported the hypotheses that Dicer and TRBP facilitate HCV infection mainly through HCV replication but not translation. The effects of Dicer and TRBP on HCV replication are not solely due to miR-122 biogenesis, and may be due to RISC loading functions in steps of miRNA gene suppression.<p> This study has set some essential groundwork for investigating potential roles of host factors in the RNAi machinery modulating HCV replication/translation and exploring novel antiviral targets.

The presence of an unpaired copy of a gene during meiosis triggers the silencing of every copy of that gene in the diploid ascus cell of Neurospora crassa, a phenomenon called Meiotic Silencing. This phenomenon has two stages: trans-sensing and meiotic silencing. If a DNA region is not detected on the opposite homologous chromosome early in meiosis (a trans-sensing failure), a signal corresponding to the unpaired region is produced that transiently silences expression of all homologous sequences. Meiotic silencing is related to RNA Silencing, a phenomenon that employs RNA-dependent RNA Polymerases (RdRPs), Argonautes, and Dicers. Dicers cleave double-stranded RNA (dsRNA) into 21-23 nucleotide RNAs. In the filamentous fungus Neurospora crassa, two RNA silencing pathways have been identified; one is active during mitosis, and the other is active during meiosis. The mitotic RNA silencing pathway, known as “quelling”, involves an RdRP (quelling-deficient-1--qde-1), an Argonaute-like protein (quelling-deficient-2--qde-2), and two Dicer-like proteins (dicer-like-1--dcl-1 and dicer-like-2--dcl-2). Previous studies in N. crassa also revealed the involvement of an RdRP (Suppressor of ascus dominance-1--Sad-1) and an Argonaute-like protein (Suppressor of meiotic silencing-2--Sms-2) in meiotic silencing, suggesting that meiotic silencing is RNA-dependent and raising the question of whether a Dicer is involved in meiotic silencing. In this work, we tested the participation in meiotic silencing of the dcl-1 gene of N. crassa, which codes for a Dicer-like protein we call Suppressor of meiotic silencing-3--Sms-3. Crosses homozygous for mutant alleles of Sms-3 are barren, indicating that the gene is also essential for sexual development. Due to this homozygous sterility, we could only test the involvement of Sms-3 in meiotic silencing in heterozygous crosses. Under these conditions, we observed suppression of the meiotic silencing which would have otherwise been induced by the presence of unpaired DNA of reporter genes. We conclude that the Dicerlike protein Sms-3 is required for both meiotic RNA silencing and sexual development.

RNA interference, or RNAi, is a cellular mechanism that describes the sequence-specific post transcriptional gene silencing observed in plants, fungi, and metazoans, facilitated by short double-stranded RNAs and microRNAs (miRNAs) with sequence complementarity to target mRNAs. Many of the regulatory mechanisms of the RNAi pathway by which these small miRNAs are first processed, from primary transcripts to precursor miRNA stemloops and then to mature miRNAs, by the multiprotein complexes Drosha and Dicer, respectively, still remain unknown. Within the miRNA biogenesis pathway, there is strong evidence pointing to the terminal loop region as an important regulatory determinant of miRNA maturation. To further elucidate the terminal loop's exerted control over miRNA processing, we propose a combined in vitro / in vivo selection experiment of a randomized pri-miRNA terminal loop library in search of an optimally processed pre-miRNA substrate. Here, we report the isolation of a premiRNA terminal loop sequence that is favorably processed by Drosha in vivo but also functions as an effective cis-inhibitor of further pre-miRNA processing by downstream Dicer. This terminal loop also demonstrated modular properties of Dicer inhibition in two different miRNAs, and should prove useful in further elucidating the mechanisms of miRNA processing in context of a newly proposed Dicer cleavage model (Gu et al. 2012). In combination, these findings may have important implications in both Drosha and Dicer's direct role in gene expression and miRNA biogenesis, the regulatory proteins that modulate their respective functions, as well as the potential development of new design rules for the more efficient processing and targeting of miRNA-based technology and RNAi therapeutics. / text

It has previously been demonstrated that lactic acid bacteria are able to influence the innate immune response of host cells. One way this can be achieved is through modulation of inflammatory cascades initiated by pattern recognition elements such as toll-like receptors. Micro RNA can also have an effect on innate immunity, and has been shown to have an influence in regulation of these pathways in immune responsive cells. However, it is yet to be determined if the interaction between lactic acid bacteria and host cells involves regulation of the RNA interference machinery involved in micro RNA biogenesis. Three of the key proteins responsible for miRNA production and activation are Argonaute 2, Dicer and Drosha. Together, these are responsible for the processing and activation of miRNA to enable post-transcriptional gene regulation. In this study we have used quantitative PCR to evaluate changes in gene expression of these enzymes in HT29 and VK2/E6E7 mucosal epithelial cells after treatment with Lactobacillus and uropathogenic bacteria. We have found that bacterial treatment downregulates gene expression of elements responsible for miRNA biogenesis, and our results showed different responses dependent on the cell line. In addition to this we have also determined stable reference genes for use in further studies involving this model. Our findings indicate that modulation of the RNAi machinery might be an important element of immune regulation by bacterial colonists.

The renin angiotensin system (RAS) is critical for the regulation of blood pressure, electrolyte/fluid, and metabolic homeostasis. Regulation of the RAS is important in the development and treatment of hypertension. As part of the rate-limiting step in a cascade of events ending in the production of angiotensin II, renin is a major regulator of the RAS. Its expression is localized to the juxtaglomerular (JG) cells of the JG apparatus where it is exquisitely located to respond to various physiological cues. Understanding the regulation of renin expression and development of the juxtaglomerular cells is critical. Two regulatory elements, the enhancer and proximal promoter, have been found to be important in controlling cell- and tissue- specific baseline expression of the renin gene. Within the enhancer is a hormone response element (HRE) which confers a high level of activity to the enhancer. Nuclear receptors that bind this element have been found to bind the HRE and regulate renin promoter transcriptional activity. We have previously characterized the role of the orphan nuclear receptor Nr2f6 as a negative regulator of renin expression that mediates its effects through the HRE. However, gel shift assays indicate that there are other transcription factors binding this element. We have identified other orphan nuclear receptors that regulate renin expression. The first, Nr2f2 acts as a negative regulator of renin promoter activity but does not appear to affect baseline expression of the endogenous renin gene. The other, Nr4a1, is a positive regulator of renin expression, but it does not appear to mediate its effects through the HRE. The transcriptional regulation of gene expression is controlled by regulatory elements separated by large distances from promoters. We and others have found that short transgenes of the human renin (hREN) locus are not sufficient to protect them from positional effects that can be exerted upon them by neighboring regulatory elements. We discovered a random truncation in a large genomic construct of the hREN gene that resulted in ubiquitous expression of renin not seen with the intact form. By locating the genomic insertion site of that transgene in the Zbtb20 gene, we found that the hREN promoter had come under control of that gene's regulatory elements. The gene downstream of renin however maintained its tissue-specific expression. We found that CCCTC-binding factor (CTCF) bound to chromatin in and around the renin locus. The presence of CTCF suggests that insulator elements are present in the renin locus, and their loss likely explains the results above. Finally, we assessed the role of microRNAs in the development of renin expressing cells in the mouse kidneys by cell-specific deletion of the processing enzyme Dicer. This resulted in reduction of renin expression and a decrease in the number of renin expressing cells in the kidney. Mice were hypotensive and had several kidney abnormalities including a hypertrophied vasculature and striped fibrosis. These results indicate that Dicer and the miRNAs it processes are critical for the development and maintenance of renin expressing cells that contribute to normal kidney development.

For more than a decade, studies focused on RNA interference (RNAi) pathway as a pivotal gene regulatory mechanism. RNAi components are attracting considerable interest due to the recent evidence demonstrating that they play a role not only in post-transcriptional regulation but also in transcriptional level. The involvement of RNAi components in heterochromatin formation and RNA Pol II processivity and alternative splicing in different organisms has been shown. Dicer protein, a highly conserved protein among kingdoms, is one of the main effectors in this pathway. There is a considerable amount of literature on Dicer’s role in the cytoplasm; however, there is still vast ambiguity concerning nuclear Dicer. More recent evidence reveals the existence of Dicer1 variants that are differentially expressed in some cancer cells. Our experiments set out to investigate one of these variants that we hypothesise is responsible for the nuclear function. We undertook genomic and biochemical approaches applied to HAP 1 cells as a model system to characterise Dicer1-s, taking advantage of a custom-made antibody in our research group. Here, as anticipated, our experiments proved that Dicer1-s is enriched in the nuclear compartment compared to full-length Dicer1, indicating that it might be a putative contributor to nuclear gene regulation activity. Unfortunately, it was not possible to establish a mutant cell line to investigate the significant nuclear function of Dicer1-s, due to the need for further optimisation of the methods used. Exploitation of previously optimised gene knock-out tools might accelerate shedding light on protein, DNA, and RNA partners, disclosing the exact nuclear mechanisms that might exhibit similar activity.

Chez la majorité des eucaryotes, les petits ARN (miRNA et siRNA) jouent des rôles essentiels au cours du développement, dans les réponses adaptatives aux stress, et dans la maintenance de la stabilité génétique. Les plantes codent quatre enzymes RNaseIII de type DICER-LIKE (DCL). DCL1, produit les miRNAs, tandis que DCL2, DCL3 et DCL4 produisent des siRNAs des tailles diverses. Les plantes codent également des enzymes appelées RNASE-THREE-LIKE (RTL) auxquelles il manque certains domaines spécifiques aux DCLs, et dont la fonction est largement inconnue.Des plantes sur-exprimant RTL1 montrent des défauts morphologiques, et n'accumulent pas les siRNAs produits par DCL2, DCL3 ou DCL4, indiquant que RTL1 est un suppresseur général des voies de siRNA chez les plantes. L’activité de RTL1 nécessite un domaine RNaseIII fonctionnel. RTL1 ne s'exprime naturellement que faiblement dans les racines, mais l'infection virale induite fortement son expression dans les feuilles, ce qui suggère que l’induction de RTL1 est une stratégie générale utilisée par les virus pour contrer la défense antivirale basée sur siRNAs. En accord avec cette hypothèse, les plantes transgéniques sur-exprimant RTL1 sont plus sensibles à l'infection par le TYMV que des plantes de type sauvage, probablement parce que RTL1 empêche la production des siRNAs dirigés contre les RNA viraux. Cependant, les plantes transgéniques sur-exprimant RTL1 ne sont pas plus sensibles à l'infection par le TCV, TVCV ou le CMV, qui codent les suppresseurs de RNA silencing (VSR) plus puissants que le TYMV. En effet, le VSR de TCV inhibe l'activité de RTL1, suggérant que l'induction de l’expression de RTL1 par les virus et l’amortissement de l’activité de RTL1 par leurs VSRs est une double stratégie permettant d’établir une infection avec succès. Des plantes sur-exprimant RTL2 ou des mutants rtl2 ne montrent aucun défaut morphologique, et ne montrent pas de changement majeur du répertoire des petits ARNs endogènes. Toutefois, la sur-expression de RTL2 augmente l’accumulation des petits ARNs exogènes dans des essais d’expression transitoire, et cette activité nécessite un domaine RNaseIII fonctionnel. Il est donc possible que RTL2 clive certains substrats pour faciliter l’action des enzymes DCL. / Small RNAs, including miRNA and siRNA, play essential regulatory roles in genome stability, development and stress responses in most eukaryotes. Plants encode four DICER-LIKE (DCL) RNaseIII enzymes. DCL1 produces miRNAs, while DCL2, DCL3 and DCL4 produce diverse size classes of siRNA. Plants also encode RNASE THREE-LIKE (RTL) enzymes that lack DCL-specific domains and whose function is largely unknown. Arabidopsis plants over-expressing RTL1 exhibit morphological defects and lack all types of small RNAs produced by DCL2, DCL3 and DCL4, indicating that RTL1 is a general suppressor of plant siRNA pathways. RTL1 activity requires a functional RNaseIII domain. RTL1 is naturally expressed only weakly in roots, but virus infection strongly induces its expression in leaves, suggesting that RTL1 induction is a general strategy used by viruses to counteract the siRNA-based plant antiviral defense. Accordingly, transgenic plants over-expressing RTL1 are more sensitive to TYMV infection than wild-type plants, likely because RTL1 prevents the production of antiviral siRNAs. However, TCV, TVCV and CMV, which encode stronger suppressors of RNA silencing (VSR) than TYMV, are insensitive to RTL1 over-expression. Indeed, TCV VSR inhibits RTL1 activity, suggesting that inducing RTL1 expression and dampening RTL1 activity is a dual strategy used by viruses to establish a successful infection. Plants over-expressing RTL2 and rtl2 mutants do not exhibit morphological defects and do not show major changes in the endogenous small RNA repertoire. However, RTL2 over-expression enhances the accumulation of exogenous siRNAs in transient assays, and this activity requires a functional RNaseIII domain. Therefore, it is possible that plant RTL2 processes certain substrates to facilitate the action of DCL enzymes.

O 4-hidroxi-2-nonenal (4-HNE) é um dos principais produtos da peroxidação lipídica, processo exacerbado no quadro de estresse oxidativo. Em função de sua alta reatividade com biomoléculas, seu acúmulo tem sido relacionado ao estabalecimento e progressão de inúmeras doenças, incluindo as cardiovasculares. Recentemente, nosso grupo identificou a interação entre 4-HNE e a proteína Dicer em coração de ratos com insuficiência cardíaca (dados não publicados). Dicer é uma RNAse importante na biogênese de microRNAs (miRNA), com papel na regulação gênica póstranscricional, de modo que alterações em sua função poderiam afetar diversos processos celulares. Tanto a interação entre o aldeído e Dicer, quanto o efeito sobre a mesma não foram descritos na literatura. Nesse contexto, o presente estudo tem como objetivo avaliar o efeito do 4-HNE na atividade e a expressão da Dicer. Nossa hipótese é que o 4-HNE afete negativamente o perfil de atividade e expressão da Dicer. Para testar essa hipótese, utilizamos o modelo animal de disfunção cardíaca induzida cirurgicamente e avaliamos: a formação de adutos de 4-HNE-proteínas, atividade e expressão de Dicer, e os níveis de miRNAs cardíacos. Em cultura celular (H9C2, MEF e HEK293), por sua vez, avaliamos o efeito agudo de 4-HNE sobre as mesmas variáveis após sua adição no meio de cultura. E, por último, utilizando a proteína recombinante, analisamos o efeito direto do aldeído sobre a estabilidade e atividade da enzima in vitro. Como esperado, em ensaios com a proteína isolada, observamos que o 4-HNE interage diretamente com a RNAse Dicer, e a formação de conjugados Dicer-4-HNE é responsável pela inibição e perda de estabilidade da proteína de forma tempo- e concentração-dependentes. No modelo animal, demonstramos um prejuízo na atividade de Dicer no coração de animais com disfunção cardíaca induzida por infarto do miocárdio, sem alteração em sua expressão, acompanhado de diminuição dos níveis da maioria dos miomiRs analisados. Notavelmente, ambos os parâmetros, assim como os níveis de adutos de 4-HNE-proteínas, foram melhorados no grupo tratado com Alda-1, agonista alostérico da enzima ALDH2 (responsável pela remoção do 4-HNE). Dessa forma, sugerimos a existência de associação entre os níveis de 4-HNE, atividade de Dicer e alteração na expressão de miRNAs no quadro de disfunção cardíaca. Consistente com os dados observados in vivo, em modelos celulares, a exposição aguda ao 4-HNE demonstrou reduzir a atividade de Dicer e afetar a via de biossíntese de miRNAs. Porém, não observamos proteção por Alda nesse modelo. Conjuntamente, nossos dados sugerem que a atividade de Dicer é modulada por 4-HNE em quadros de estresse agudo e crônico de aldeídos. Contudo, mais estudos são necessários a fim de elucidar o mecanismo pelo qual essa modulação ocorre. Visto que o acúmulo de 4-HNE e a desregulação na biogênese de miRNAs tem sido associados ao desenvolvimento de patologias, o estudo da interação entre Dicer e o aldeído é importante na compreensão dessas doenças e planejamento de novas estratégias terapêuticas. / 4-hydroxy-2-nonenal (4-HNE) is a major by-product of lipid peroxidation, a process that is exacerbated under oxidative stress conditions. This aldehyde is a very reactive molecule associated with the establishment and progression of many diseases, including cardiovascular diseases. We recently found using proteomics that 4-HNE directly targets Dicer in failing hearts, a critical enzyme for miRNA biology (unpublished data). Neither the aldehyde-Dicer adduction, nor its effect on protein stability and activity has been previously reported. Therefore, this study aimed to fill this gap by further investigating 4-HNE-Dicer interaction and characterizing its effect on Dicer profile. We hypothesize that 4-HNE will make adducts with Dicer and compromise its function and levels. Using an animal model of cardiac dysfunction, we evaluated the following parameters: levels of 4-HNE adducted proteins, Dicer levels and activity, and the levels of heart specific miRNAs (myomiRs). The same variables were analyzed in distinct cellular models (H9C2, MEF, HEK293) after acute exposure to 4-HNE. Additionally, we synthetized recombinant Dicer, and protein function and stability were assessed in vitro. As expected, the experiments with recombinant protein revealed that 4-HNE directly interacts with Dicer, and the formation of 4-HNE-DICER adduct causes loss of Dicer cleavage activity and stability in a time- and concentration-dependent manner. Regarding the animal model, Dicer activity, but not protein levels, dropped in failing hearts, which was paralleled by a reduction of mature miRNA levels. Of interest, animals with cardiac dysfunction chronically treated with a small molecule activator of aldehyde dehydrogenase 2 (ALDH2), termed Alda-1, displayed an elevated cardiac Dicer activity and mature miRNA levels compared with vehicle-treated animals. ALDH2 is the mains enzyme responsible for 4-HNE clearance. In this context, this study points out a potential connection among 4-HNE levels, Dicer activity and myomiR levels in cardiac dysfunction. Consistent with our in vivo data, cells acutely exposed to 4-HNE showed an increase in 4-HNE-protein adducts followed by a reduction in Dicer activity and changes in miRNA biosynthesis. However, Alda showed no protective effect in the latter model. Taken together, our findings using animal and cellular models suggest that Dicer activity is impaired in chronic (cardiac dysfunction) and acute aldehyde stress conditions. However, the molecular mechanisms involved in this response are still unclear. As both 4-HNE accumulation and microRNAs have been linked to innumerous pathologies, clarifying the modulation of Dicer activity under such conditions will certainly contribute to a better understanding the diseases and future therapeutic strategies.