Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA

Two small RNAs regulate the timing of Caenorhabditis elegans development(1,2). Transition from the first to the second larval stage fates requires the 22-nucleotide lin-4 RNA(1,3,4), and transition from late larval to adult cell fates requires the 21-nucleotide let-7 RNA 2. The lin-4 and let-7 RNA genes are not homologous to each other, but are each complementary to sequences in the 3' untranslated regions of a set of protein-coding target genes that are normally negatively regulated by the RNAs1,2,5,6. Here we have detected let-7 RNAs of similar to 21 nucleotides in samples from a wide range of animal species, including vertebrate, ascidian, hemichordate, mollusc, annelid and arthropod, but not in RNAs from several cnidarian and poriferan species, Saccharomyces cerevisiae, Escherichia coli or Arabidopsis. We did not detect lin-4 RNA in these species. We found that let-7 temporal regulation is also conserved: let-7 RNA expression is first detected at late larval stages in C. elegans and Drosophila, at 48 hours after fertilization in zebrafish, and in adult stages of annelids and molluscs. The let-7 regulatory RNA may control late temporal transitions during development across animal phylogeny.

The Lin28/let-7 axis regulates glucose metabolism.

The let-7 tumor suppressor microRNAs are known for their regulation of oncogenes, while the RNA-binding proteins Lin28a/b promote malignancy by inhibiting let-7 biogenesis. We have uncovered unexpected roles for the Lin28/let-7 pathway in regulating metabolism. When overexpressed in mice, both Lin28a and LIN28B promote an insulin-sensitized state that resists high-fat-diet induced diabetes. Conversely, muscle-specific loss of Lin28a or overexpression of let-7 results in insulin resistance and impaired glucose tolerance. These phenomena occur, in part, through the let-7-mediated repression of multiple components of the insulin-PI3K-mTOR pathway, including IGF1R, INSR, and IRS2. In addition, the mTOR inhibitor, rapamycin, abrogates Lin28a-mediated insulin sensitivity and enhanced glucose uptake. Moreover, let-7 targets are enriched for genes containing SNPs associated with type 2 diabetes and control of fasting glucose in human genome-wide association studies. These data establish the Lin28/let-7 pathway as a central regulator of mammalian glucose metabolism.

IMP2 provides an alternative to LIN28B for LET-7 target gene protection and glioma stem cell maintenance

Glioblastoma multiforme (GBM) is the most frequent and lethal primary brain tumor in adults. Accumulating evidence suggests that tumors comprise a hierarchical organization that is, at least partially, not genetically driven. Cells that reside at the apex of this hierarchy are commonly referred to as cancer stem cells (CSCs) and are believed to largely contribute to recurrence and therapeutic failure. Although the complexity of epigenetic regulation of the genome precludes prediction as to which epigenetic changes dominate CSC specification in different cancer types, the ability of microRNAs (miRNAs) to fine-tune expression of entire gene networks places them among prime candidates for establishing CSC properties. In this study we characterized the miRNA expression profile of primary GBM grown either under conditions that enrich for GSCs or their differentiated non-tumorigenic progeny (DGCs). Although, we identified a subset of miRNAs that was strongly differentially expressed between GSCs and DGCs, we observed that in GSCs both let-7 and, paradoxically, their target genes are highly expressed, suggesting protection against let-7 action. Using PAR-CLIP we show that insulin-like growth factor-2 mRNA-binding protein 2 (IMP2) provides a mechanism for let-7 target gene protection that represents an alternative to LIN28A/B, which abrogates let-7 biogenesis in normal embryonic and certain malignant stem cells. By direct binding to miRNA recognition elements, IMP2 protects its targets from let-7 mediated decay. Importantly, depletion of IMP2 in GSCs strongly impairs their self- renewal properties and tumorigenicity in vivo, a phenotype that can be rescued by expression of LIN28B, suggesting that IMP2 mainly contributes to GSC maintenance by protecting let-7 target genes from silencing. Using mouse models, we show that depletion of IMP2 in neural stem cells (NSCs) induces let-7 target gene down-regulation, impairs their clonogenic capacity, and affects differentiation. Taken together, our observations describe a novel regulatory function of IMP2 in the let-7 axis whereby it supports GSC and NSC specification. Résumé (Français) Le glioblastome (GBM) est la tumeur primaire maligne du cerveau la plus fréquente. De nombreuses études ont démontré l'existence d'une organisation hiérarchique des cellules cancéreuses liée à des mécanismes épigénétiques. Les cellules qui se trouvent au sommet de cette hiérarchie sont appelées cellules souches cancéreuses (CSC), et contribuent à l'échec thérapeutique. Bien que la complexité des régulateurs épigénétiques permette difficilement de prédire quel mécanisme contribue le plus aux propriétés des CSC, la capacité des microRNAs (miRNAs) de réguler des réseaux entiers de gènes, les placent comme des candidats de premiers choix. Ici, nous avons caractérisé le profil d'expression des miRNAs dans des tumeurs primaires de GBM cultivées dans des conditions qui enrichissent soit pour les CSC, soit pour leur contrepartie de cellules cancéreuses différences (CCD). De manière surprenante et paradoxale la famille de miRNA let-7 et leurs gènes cibles étaient hautement exprimés dans les CSC, suggérant un mécanisme de protection contre l'action des let-7. Avec l'aide de la technologie PAR-CLIP, nous démontrons que la protéine IMP2, protège les mRNAs de l'action des let-7 et représente une alternative à Lin28A/B, qui d'ordinaire réprime fortement la maturation des let-7 dans les cellules souches embryonnaires et divers cancers. En se liant à la région ciblée par les let-7, IMP2 protège ses transcrits de l'action de cette classe de microRNA qui est tumoro-supressive. La déplétion d'IMP2 dans des CSC de GBM réduit fortement leur clonogénicité in vitro et leur tumorigénicité in vivo. Ceci peut être reversé en introduisant Lin28B dans des CSC de GBM, suggérant qu'IMP2 exerce ses fonctions pro-tumorigéniques en modulant l'axe let-7. Avec l'aide de modèles murins, nous observons que la déplétion de IMP2 dans les cellules souches neurales (CSN) induit une baisse de leur clonogénicité et des cibles des miRNAs let-7, suggérant une conservation de ce mécanisme entre les CSC de GBM et les CSN. En résumé, nos observations définissent une nouvelle fonction de IMP2 dans l'axe let-7 par lequel il contribue au maintien des propriétés des CSC et des CSN.

Effects of let-7 microRNA on Cell Growth and Differentiation of Papillary Thyroid Cancer

Papillary thyroid carcinoma (PTC) is the most common endocrine malignancy and RET/PTC rearrangements represent key genetic events frequently associated to this cancer, enhancing proliferation and dedifferentiation by activation of the RET/PTC-RAS-BRAF-mitogen-activated protein kinase (MAPK) pathway. Recently, let-7 microRNA was found to reduce RAS levels in lung cancer, acting as a tumor suppressor gene. Here, we report that RET/PTC3 oncogenic activation in PCCL3 rat thyroid cells markedly reduces let-7f expression. Moreover, stable transfection of let-7 microRNA in TPC-1 cells, which harbor RET/PTC1 rearrangement, inhibits MAPK activation. As a result, let-7f was capable of reducing TPC-1 cell growth, and this might be explained, at least in part, by decreased messenger RNA (mRNA) expression of cell cycle stimulators such as MYC and CCND1 (cyclin D1) and increased P21 cell cycle inhibitor mRNA. In addition, let-7 enhanced transcriptional expression of molecular markers of thyroid differentiation such as TITF1 and TG. Thus, reduced expression of let-7f might be an essential molecular event in RET/PTC malignant transformation. Moreover, let-7f effects on thyroid growth and differentiation might attenuate neoplastic process of RET/PTC papillary thyroid oncogenesis through impairment of MAPK signaling pathway activation. This is the first functional demonstration of an association of let-7 with thyroid cancer cell growth and differentiation.

ROLE OF MICRORNA LET-7 IN PANCREATIC BETA CELLS

MicroRNAs (miRNAs) are small non-coding RNAs that inhibit gene expression at transcriptional or post-transcriptional level. Let-7 family is among the first identified human miRNAs and regulates multiple cellular processes including glucose metabolism in multiple organs. It has been reported that overexpression of let-7 resulted in insulin resistance and impaired glucose tolerance through repressing insulin signaling pathway in both muscle and liver. However, the role and mechanism underlying let-7 function in pancreatic beta-cells have yet to be elucidated. Let-7 family contains nine members, which poses a significant challenge in complete deletion of this miRNA family. To study the function of let-7 and to overcome the functional redundancies of various let-7 members in pancreatic beta-cells, the highly expressed let-7a and let-7b were blocked simultaneously using short tandem target mimic (STTM) approach developed in our laboratory. Introducing STTM-let7 into beta-cells markedly increased the expression of Caspase 3, a direct target of let-7, confirming a sufficient functional knockdown of let-7a/b by STTM-let7. STTM-let7 enhanced apoptotic cell death induced by cytokine, indicating that let-7a/b is able to protect from apoptosis through attenuating Caspase 3 expression in pancreatic beta-cells. In contrast to the previous observation that let-7 silencing increases insulin signaling in muscle and liver, inhibition of let-7 with STTM-let7 significantly repressed glucose-stimulated insulin signaling in pancreatic beta-cells, leading to impaired insulin secretion and reduced beta-cell proliferation. Taken together, an appropriate level of let-7 is essential in maintaining beta-cell function and viability. Dysregulation of let-7 may contribute to the pathogenesis of type 2 diabetes.

Distinct microRNA Expression Profile in Prostate Cancer Patients with Early Clinical Failure and the Impact of let-7 as Prognostic Marker in High-Risk Prostate Cancer

Background The identification of additional prognostic markers to improve risk stratification and to avoid overtreatment is one of the most urgent clinical needs in prostate cancer (PCa). MicroRNAs, being important regulators of gene expression, are promising biomarkers in various cancer entities, though the impact as prognostic predictors in PCa is poorly understood. The aim of this study was to identify specific miRNAs as potential prognostic markers in high-risk PCa and to validate their clinical impact. Methodology and Principal Findings We performed miRNA-microarray analysis in a high-risk PCa study group selected by their clinical outcome (clinical progression free survival (CPFS) vs. clinical failure (CF)). We identified seven candidate miRNAs (let-7a/b/c, miR-515-3p/5p, -181b, -146b, and -361) that showed differential expression between both groups. Further qRT-PCR analysis revealed down-regulation of members of the let-7 family in the majority of a large, well-characterized high-risk PCa cohort (n = 98). Expression of let-7a/b/and -c was correlated to clinical outcome parameters of this group. While let-7a showed no association or correlation with clinical relevant data, let-7b and let-7c were associated with CF in PCa patients and functioned partially as independent prognostic marker. Validation of the data using an independent high-risk study cohort revealed that let-7b, but not let-7c, has impact as an independent prognostic marker for BCR and CF. Furthermore, we identified HMGA1, a non-histone protein, as a new target of let-7b and found correlation of let-7b down-regulation with HMGA1 over-expression in primary PCa samples. Conclusion Our findings define a distinct miRNA expression profile in PCa cases with early CF and identified let-7b as prognostic biomarker in high-risk PCa. This study highlights the importance of let-7b as tumor suppressor miRNA in high-risk PCa and presents a basis to improve individual therapy for high-risk PCa patients.

PreImplantation factor promotes neuroprotection by targeting microRNA let-7

Dysfunction and loss of neurons are the major characteristics of CNS disorders that include stroke, multiple sclerosis, and Alzheimer's disease. Activation of the Toll-like receptor 7 by extracellular microRNA let-7, a highly expressed microRNA in the CNS, induces neuronal cell death. Let-7 released from injured neurons and immune cells acts on neighboring cells, exacerbating CNS damage. Here we show that a synthetic peptide analogous to the mammalian PreImplantation factor (PIF) secreted by developing embryos and which is present in the maternal circulation during pregnancy inhibits the biogenesis of let-7 in both neuronal and immune cells of the mouse. The synthetic peptide, sPIF, destabilizes KH-type splicing regulatory protein (KSRP), a key microRNA-processing protein, in a Toll-like receptor 4 (TLR4)-dependent manner, leading to decreased production of let-7. Furthermore, s.c. administration of sPIF into neonatal rats following hypoxic-ischemic brain injury robustly rescued cortical volume and number of neurons and decreased the detrimental glial response, as is consistent with diminished levels of KSRP and let-7 in sPIF-treated brains. Our results reveal a previously unexpected mechanism of action of PIF and underscore the potential clinical utility of sPIF in treating hypoxic-ischemic brain damage. The newly identified PIF/TLR4/KSRP/let-7 regulatory axis also may operate during embryo implantation and development.

The H19/let-7 double-negative feedback loop contributes to glucose metabolism in muscle cells

The H19 lncRNA has been implicated in development and growth control and is associated with human genetic disorders and cancer. Acting as a molecular sponge, H19 inhibits microRNA (miRNA) let-7. Here we report that H19 is significantly decreased in muscle of human subjects with type-2 diabetes and insulin resistant rodents. This decrease leads to increased bioavailability of let-7, causing diminished expression of let-7 targets, which is recapitulated in vitro where H19 depletion results in impaired insulin signaling and decreased glucose uptake. Furthermore, acute hyperinsulinemia downregulates H19, a phenomenon that occurs through PI3K/AKT-dependent phosphorylation of the miRNA processing factor KSRP, which promotes biogenesis of let-7 and its mediated H19 destabilization. Our results reveal a previously undescribed double-negative feedback loop between sponge lncRNA and target miRNA that contributes to glucose regulation in muscle cells.

Let-7a Is a Direct EWS-FLI-1 Target Implicated in Ewing's Sarcoma Development.

Ewing's sarcoma family tumors (ESFT) are the second most common bone malignancy in children and young adults, characterized by unique chromosomal translocations that in 85% of cases lead to expression of the EWS-FLI-1 fusion protein. EWS-FLI-1 functions as an aberrant transcription factor that can both induce and suppress members of its target gene repertoire. We have recently demonstrated that EWS-FLI-1 can alter microRNA (miRNA) expression and that miRNA145 is a direct EWS-FLI-1 target whose suppression is implicated in ESFT development. Here, we use miRNA arrays to compare the global miRNA expression profile of human mesenchymal stem cells (MSC) and ESFT cell lines, and show that ESFT display a distinct miRNA signature that includes induction of the oncogenic miRNA 17-92 cluster and repression of the tumor suppressor let-7 family. We demonstrate that direct repression of let-7a by EWS-FLI-1 participates in the tumorigenic potential of ESFT cells in vivo. The mechanism whereby let-7a expression regulates ESFT growth is shown to be mediated by its target gene HMGA2, as let-7a overexpression and HMGA2 repression both block ESFT cell tumorigenicity. Consistent with these observations, systemic delivery of synthetic let-7a into ESFT-bearing mice restored its expression in tumor cells, decreased HMGA2 expression levels and resulted in ESFT growth inhibition in vivo. Our observations provide evidence that deregulation of let-7a target gene expression participates in ESFT development and identify let-7a as promising new therapeutic target for one of the most aggressive pediatric malignancies.

Caractérisation de l’interaction entre la protéine Lin28 et le précurseur du microARN let-7g

La régulation de l’expression des gènes est ce qui permet à nos cellules de s’adapter à leur environnement, de combattre les infections ou, plus généralement, de produire la quantité exacte de protéine nécessaire pour répondre à un besoin spécifique. Parmi les joueurs les plus importants dans cette régulation de l’expression des gènes on retrouve les microARN (miARN). Ces petits ARN de 22 nucléotides sont présents chez la majorité des espèces multicellulaires et sont responsables du contrôle direct de plus de 30% des gènes exprimant des protéines chez les vertébrés. La famille de miARN lethal-7 (let-7) est composée de miARN parmi les plus connus et ayant des fonctions cruciales pour la cellule. La régulation du niveau des miARN let-7 est essentielle au bon développement cellulaire. La biogenèse de ces miARN, du transcrit primaire jusqu’à leur forme mature, est régulée principalement par Lin28, une protéine pluripotente très conservée. Cette protéine est composée d’un domaine cold shock (CSD) et de deux domaines de liaison au zinc. C’est grâce à ces domaines de liaison à l’ARN que Lin28 peut lier et inhiber la maturation des miARN let-7. L’objectif de cette thèse est de caractériser l’interaction entre Lin28 et le microARN précurseur let-7g afin de mieux comprendre le rôle de cette protéine dans l’inhibition de la biogenèse du miARN. À l’aide de techniques biochimiques et biophysiques, nous avons d’abord défini les principaux déterminants de l’interaction entre Lin28 et la boucle terminale du miARN précurseur let-7g (TL-let-7g). Nous avons conclu que le domaine C-terminal de Lin28, composé d’un motif riche en lysines et arginines ainsi que de deux motifs de liaison au zinc, permet à la protéine de lier spécifiquement et avec haute affinité un renflement riche en guanine conservé chez les précurseurs de la famille let-7. Aussi, parce que la séquence et la spécificité de liaison à l’ARN de ce domaine C-terminal sont semblables à celles de la protéine NCp7 du VIH, nous avons défini ce dernier comme le domaine NCp7-like de Lin28. Par la suite, nous avons caractérisé la multimérisation de trois protéines Lin28 sur la boucle terminale de pre-let-7g. Ceci a permis de réconcilier d’apparentes contradictions retrouvées dans la littérature actuelle concernant les sites de liaison de Lin28 lors de sa liaison aux miARN précurseurs. Nous avons identifié trois sites de liaison à haute affinité sur TL-let-7g qui sont liés dans un ordre précis par trois protéines Lin28. Lors de la formation du complexe multimérique, le CSD permet une déstabilisation de l’ARN, ce qui rend accessible plusieurs sites de liaison. Le domaine NCp7-like permet plutôt un assemblage ordonné de la protéine et facilite la liaison initiale de cette dernière. Ces nouveaux résultats rendent possible la mise au point d’un nouveau modèle de l’interaction entre Lin28 et le miARN précurseur let-7g. En conclusion, les études réalisées dans cette thèse apportent une meilleure compréhension des mécanismes moléculaires impliqués dans la régulation post-transcriptionnelle d’une importante famille de miARN et permettront de guider les futures études dans le domaine de recherche en pleine effervescence qu’est celui de la biogenèse des miARN.

MicroRNAs in Ewing's Sarcoma

Les tumeurs de la famille du sarcome d'Ewing (ESFTs) sont les deuxièmes plus fréquentes formes de cancer de l'os chez l'enfant et l'adolescent. Le gène de fusion EWS-FLI1 est associé à 85-90% des ESFTs. Ce cancer a probablement pour origine des cellules souches mésenchymateuses (MSCs). Il a en effet été démontré que les MSCs pédiatriques (hpMSCs) sont particulièrement permissives pour le gène de fusion EWS-FLI1 et que celui-ci induit des gènes de cellules souches embryonnaires. Ceci génère une sous-population de cellules présentant des caractéristiques de cellules souches cancéreuses de sarcome d'Ewing (ESFT CSCs) in vitro. Ces cellules reprogrammées n'ont pas de potentiel tumorigénique et un certain nombre de microARN ne sont pas réprimés ou exprimés comme dans un sarcome d'Ewing primaire et sa sous-population de CSCs. Parmi ces microARN on trouve en particulier les membres de la famille let-7 qui jouent un rôle clé dans le contrôle de l'état de différenciation des cellules et régulent de nombreux oncogènes. De plus, leur répression serait capable de favoriser la tumorigénèse. Tous les membres de la famille des microARNs let-7 ont un régulateur commun, la protéine lin-28, qui exerce notamment son action en bloquant la maturation de ces microARNs. Dans ce travail, il s'agira d'évaluer si la co-expression de EWS-FLI1 et de lin-28 dans des hpMSCs permet de créer une sous-population de cellules présentant les caractéristiques de ESFT CSCs. Nous évaluerons l'effet de lin-28 sur les membres de la famille des let7 dans les hpMSCs et apprécierons le potentiel tumorigénique in vivo des hpMSCs exprimant EWS-FLI1 et lin-28. L'outil « Targetscan » est un logiciel qui permet de prédire les cibles des microARN en analysant leur séquence et en la comparant à l'ARN messager 3' non transcrit. Pour les microARN de la famille des let-7, cet outil identifie des gènes cibles potentiels qui jouent un rôle important dans le sarcome d'Ewing. Nous évaluerons si ces protéines sont en effet régulées de façon let-7 dépendante et les conséquences sur la pathogénèse des ESFTs.

HelF, a suppressor of RNAi mediated gene silencing in Dictyostelium discoideum

RNA interference (RNAi) is a recently discovered process, in which double stranded RNA (dsRNA) triggers the homology-dependant degradation of cognate messenger RNA (mRNA). In a search for new components of the RNAi machinery in Dictyostelium, a new gene was identified, which was called helF. HelF is a putative RNA helicase, which shows a high homology to the helicase domain of Dicer, to the helicase domain of Dictyostelium RdRP and to the C. elegans gene drh-1, that codes for a dicer related DExH-box RNA helicase, which is required for RNAi. The aim of the present Ph.D. work was to investigate the role of HelF in PTGS, either induced by RNAi or asRNA. A genomic disruption of the helF gene was performed, which resulted in a distinct mutant morphology in late development. The cellular localization of the protein was elucidated by creating a HelF-GFP fusion protein, which was found to be localized in speckles in the nucleus. The involvement of HelF in the RNAi mechanism was studied. For this purpose, RNAi was induced by transformation of RNAi hairpin constructs against four endogenous genes in wild type and HelF- cells. The silencing efficiency was strongly enhanced in the HelF K.O. strain in comparison with the wild type. One gene, which could not be silenced in the wild type background, was successfully silenced in HelF-. When the helF gene was disrupted in a secondary transformation in a non-silenced strain, the silencing efficiency was strongly improved, a phenomenon named here “retrosilencing”. Transcriptional run-on experiments revealed that the enhanced gene silencing in HelF- was a posttranscriptional event, and that the silencing efficiency depended on the transcription levels of hairpin RNAs. In HelF-, the threshold level of hairpin transcription required for efficient silencing was dramatically lowered. The RNAi-mediated silencing was accompanied by the production of siRNAs; however, their amount did not depend on the level of hairpin transcription. These results indicated that HelF is a natural suppressor of RNAi in Dictyostelium. In contrast, asRNA mediated gene silencing was not enhanced in the HelF K.O, as shown for three tested genes. These results confirmed previous observations (H. Martens and W. Nellen, unpublished) that although similar, RNAi and asRNA mediated gene silencing mechanisms differ in their requirements for specific proteins. In order to characterize the function of the HelF protein on a molecular level and to study its interactions with other RNAi components, in vitro experiments were performed. Besides the DEAH-helicase domain, HelF contains a double-stranded RNA binding domain (dsRBD) at its N-terminus, which showed high similarity to the dsRBD domain of Dicer A from Dictyostelium. The ability of the recombinant dsRBDs from HelF and Dicer A to bind dsRNA was examined and compared. It was shown by gel-shift assays that both HelF-dsRBD and Dicer-dsRBD could bind directly to long dsRNAs. However, HelF-dsRBD bound more efficiently to dsRNA with imperfect matches than to perfect dsRNA. Both dsRBDs bound specifically to a pre-miRNA substrate (pre-let-7). The results suggested that most probably there were two binding sites for the proteins on the pre-miRNA substrate. Moreover, it was shown that HelF-dsRBD and Dicer-dsRBD have siRNA-binding activity. The affinities of the two dsRBDs to the pre-let-7 substrate were also examined by plasmon surface resonance analyses, which revealed a 9-fold higher binding affinity of the Dicer-dsRBD to pre-let-7 compared to that of the HelF-dsRBD. The binding of HelF-dsRBD to the pre-let-7 was impaired in the presence of Mg2+, while the Dicer-dsRBD interaction with pre-let-7 was not influenced by the presence of Mg2+. The results obtained in this thesis can be used to postulate a model for HelF function. In this, HelF acts as a nuclear suppressor of RNAi in wild type cells by recognition and binding of dsRNA substrates. The protein might act as a surveillance system to avoid RNAi initiation by fortuitous dsRNA formation or low abundance of dsRNA trigger. If the protein acts as an RNA helicase, it could unwind fold-back structures in the nucleus and thus lead to decreased RNAi efficiency. A knock-out of HelF would result in initiation of the RNAi pathway even by low levels of dsRNA. The exact molecular function of the protein in the RNAi mechanism still has to be elucidated. RNA interferenz (RNAi) ist ein in jüngster Zeit entdeckter Mechanismus, bei dem doppelsträngige RNA Moleküle (dsRNA) eine Homologie-abhängige Degradation einer verwandten messenger-RNA (mRNA) auslösen. Auf der Suche nach neuen Komponenten der RNAi-Maschinerie in Dictyostelium konnte ein neues Gen (helF) identifiziert werden. HelF ist eine putative RNA-Helikase mit einer hohen Homologie zur Helikasedomäne der bekannten Dicerproteine, der Helikasedomäne der Dictyostelium RdRP und zu dem C. elegans Gen drh-1, welches für eine Dicer-bezogene DExH-box RNA Helikase codiert, die am RNAi-Mechanismus beteiligt ist. Das Ziel dieser Arbeit war es, die Funktion von HelF im Zusammenhang des RNAi oder asRNA induzierten PTGS zu untersuchen. Es wurde eine Unterbrechung des helF-Gens auf genomischer Ebene (K.O.) vorgenommen, was bei den Mutanten zu einer veränderten Morphologie in der späten Entwicklung führte. Die Lokalisation des Proteins in der Zelle konnte mit Hilfe einer GFP-Fusion analysiert werden und kleinen Bereichen innerhalb des Nukleus zugewiesen werden. Im Weiteren wurde der Einfluss von HelF auf den RNAi-Mechanismus untersucht. Zu diesem Zweck wurde RNAi durch Einbringen von RNAi Hairpin-Konstrukten gegen vier endogene Gene im Wiltypstamm und der HelF--Mutante induziert. Im Vergleich zum Wildtypstamm konnte im HelF--Mutantenstamm eine stark erhöhte „Silencing“-Effizienz nachgewiesen werden. Ein Gen, welches nach RNAi Initiation im Wildtypstamm unverändert blieb, konnte im HelF--Mutantenstamm erfolgreich stillgelegt werden. Durch sekundäres Einführen einer Gendisruption im helF-Locus in einen Stamm, in welchem ein Gen nicht stillgelegt werden konnte, wurde die Effizienz des Stilllegens deutlich erhöht. Dieses Phänomen wurde hier erstmals als „Retrosilencing“ beschrieben. Mit Hilfe von transkriptionellen run-on Experimenten konnte belegt werden, dass es sich bei dieser erhöhten Stilllegungseffizienz um ein posttranskriptionelles Ereignis handelte, wobei die Stillegungseffizienz von der Transkriptionsstärke der Hairpin RNAs abhängt. Für die HelF--Mutanten konnte gezeigt werden, dass der Schwellenwert zum Auslösen eines effizienten Stillegens dramatisch abgesenkt war. Obwohl die RNAi-vermittelte Genstilllegung immer mit der Produktion von siRNAs einhergeht, war die Menge der siRNAs nicht abhängig von dem Expressionsniveau des Hairpin-Konstruktes. Diese Ergebnisse legen nahe, dass es sich bei der HelF um einen natürlichen Suppressor des RNAi-Mechanismus in Dictyostelium handelt. Im Gegensatz hierzu war die as-vermittelte Stilllegung von drei untersuchten Genen im HelF-K.O. im Vergleich zum Wildyp unverändert. Diese Ergebnisse bestätigten frühere Beobachtungen (H. Martens und W. Nellen, unveröffentlicht), wonach die Mechanismen für RNAi und asRNA-vermittelte Genstilllegung unterschiedliche spezifische Proteine benötigen. Um die Funktion des HelF-Proteins auf der molekularen Ebene genauer zu charakterisieren und die Interaktion mit anderen RNAi-Komponenten zu untersuchen, wurden in vitro Versuche durchgeführt. Das HelF-Protein enthält, neben der DEAH-Helikase-Domäne eine N-terminale Doppelstrang RNA bindende Domäne (dsRBD) mit einer hohen Ähnlichkeit zu der dsRBD des Dicer A aus Dictyostelium. Die dsRNA-Bindungsaktivität der beiden dsRBDs aus HelF und Dicer A wurde analysiert und verglichen. Es konnte mithilfe von Gel-Retardationsanalysen gezeigt werden, dass sowohl HelF-dsRBD als auch Dicer-dsRBD direkt an lange dsRNAs binden können. Hierbei zeigte sich, dass die HelF-dsRBD eine höhere Affinität zu einem imperfekten RNA-Doppelstrang besitzt, als zu einer perfekt gepaarten dsRNA. Für beide dsRBDs konnte eine spezifische Bindung an ein pre-miRNA Substrat nachgewiesen werden (pre-let-7). Dieses Ergebnis legt nah, dass es zwei Bindestellen für die Proteine auf dem pre-miRNA Substrat gibt. Überdies hinaus konnte gezeigt werden, dass die dsRBDs beider Proteine eine siRNA bindende Aktivität besitzen. Die Affinität beider dsRBDs an das pre-let-7 Substrat wurde weiterhin mit Hilfe der Plasmon Oberflächen Resonanz untersucht. Hierbei konnte eine 9-fach höhere Bindeaffinität der Dicer-dsRBD im Vergleich zur HelF-dsRBD nachgewiesen werden. Während die Bindung der HelF-dsRBD an das pre-let-7 durch die Anwesenheit von Mg2+ beeinträchtigt war, zeigte sich kein Einfluß von Mg2+ auf das Bindeverhalten der Dicer-dsRBD. Mit Hilfe der in dieser Arbeit gewonnen Ergebnisse lässt sich ein Model für die Funktion von HelF postulieren. In diesem Model wirkt HelF durch Erkennen und Binden von dsRNA Substraten als Suppressor von der RNAi im Kern. Das Protein kann als Überwachungsystem gegen eine irrtümliche Auslösung von RNAi wirken, die durch zufällige dsRNA Faltungen oder eine zu geringe Häufigkeit der siRNAs hervorgerufen sein könnte. Falls das Protein eine Helikase-Aktivität besitzt, könnte es rückgefaltete RNA Strukturen im Kern auflösen, was sich in einer verringerten RNAi-Effizienz wiederspiegelt. Durch Ausschalten des helF-Gens würde nach diesem Modell eine erfolgreiche Auslösung von RNAi schon bei sehr geringer Mengen an dsRNA möglich werden. Das Modell erlaubt, die exakte molekulare Funktion des HelF-Proteins im RNAi-Mechanismus weiter zu untersuchen.

Artificial micro RNA system in Dictyostelium discoideum

Dictyostelium discoideum is a social amoeba that serves as a model system for RNA interference and related mechanisms. Its position between plants and animals enables evolutionary snapshot of mechanisms and protein machinery involved in investigated subjects. MiRNAs are small regulatory RNAs that are evolutionary conserved and present in animals, plants, viruses and some prokaryotes. They have roles in development, cell growth and differentiation, apoptosis and their miss-regulation is associated with many diseases such as cancer, neurodegenerative disorders and diabetes. Recently, through sequencing of DNA libraries miRNAs have been discovered in D. discoideum. In this work, it has been shown that heterologues miRNA let-7 can be expressed and processed in D. discoideum. Expression of let-7 miRNA in social amoeba resulted in a strong developmental phenotype suggesting an overload of the processing/silencing system or/and endogenous targets. The various effects on prel-7 strain have been observed and characterized, serving as a background for postulation of miRNA roles. An artificial miRNA system has been established and imposed to D. discoideum, showing that miRNAs in Dictyostelium could mediate gene expression on the level of mRNA stability and on the posttranscriptional level. Furthermore, presence of translational inhibition as a type of gene control was shown for the first time in this organism. Due to it new structures representing co-localities of miRNA and target mRNA have been detected. Taken together, this work shows functional artificial miRNA system and postulates roles of endogenous small RNA in social amoeba.

Absence of Functional LIN28B Mutations in a Large Cohort of Patients with Idiopathic Central Precocious Puberty

Aim: To investigate LIN28B gene variants in children with idiopathic central precocious puberty (CPP). Patients and Methods: We studied 178 Brazilian children with CPP (171 girls, 16.8% familial cases). A large multiethnic group (1,599 subjects; Multiethnic Cohort, MEC) was used as control. DNA analysis and biochemical in vitro studies were performed. Results: A heterozygous LIN28B variant, p. H199R, was identified in a girl who developed CPP at 5.2 years. This variant was absent in 310 Brazilian control individuals, but it was found in the same allele frequency in women from the MEC cohort, independent of the age of menarche. Functional studies revealed that when ectopically expressed in cells, the mutant protein was capable of binding pre-let-7 microRNA and inhibiting let-7 expression to the same extent as wild-type Lin28B protein. Other rare LIN28B variants (p.P173P, c.198+32_33delCT, g.9575731A>C and c.-11C>T) were identified in CPP patients and controls. Therefore, no functional mutation was identified. Conclusion: In vitro studies revealed that the rare LIN28B p.H199R variant identified in a girl with CPP does not affect the Lin28B function in the regulation of let-7 expression. Although LIN28B SNPs were associated with normal pubertal timing, rare variations in this gene do not seem to be commonly involved in the molecular pathogenesis of CPP. Copyright (C) 2012 S. Karger AG, Basel

Mammalian miRNA RISC recruits CAF1 and PABP to affect PABP-dependent deadenylation.

MicroRNAs (miRNAs) inhibit mRNA expression in general by base pairing to the 3'UTR of target mRNAs and consequently inhibiting translation and/or initiating poly(A) tail deadenylation and mRNA destabilization. Here we examine the mechanism and kinetics of miRNA-mediated deadenylation in mouse Krebs-2 ascites extract. We demonstrate that miRNA-mediated mRNA deadenylation occurs subsequent to initial translational inhibition, indicating a two-step mechanism of miRNA action, which serves to consolidate repression. We show that a let-7 miRNA-loaded RNA-induced silencing complex (miRISC) interacts with the poly(A)-binding protein (PABP) and the CAF1 and CCR4 deadenylases. In addition, we demonstrate that miRNA-mediated deadenylation is dependent upon CAF1 activity and PABP, which serves as a bona fide miRNA coactivator. Importantly, we present evidence that GW182, a core component of the miRISC, directly interacts with PABP via its C-terminal region and that this interaction is required for miRNA-mediated deadenylation.