Design of small molecule-responsive microRNAs based on structural requirements for Drosha processing

MicroRNAs (miRNAs) are prevalent regulatory RNAs that mediate gene silencing and play key roles in diverse cellular processes. While synthetic RNA-based regulatory systems that integrate regulatory and sensing functions have been demonstrated, the lack of detail on miRNA structure–function relationships has limited the development of integrated control systems based on miRNA silencing. Using an elucidated relationship between Drosha processing and the single-stranded nature of the miRNA basal segments, we developed a strategy for designing ligand-responsive miRNAs. We demonstrate that ligand binding to an aptamer integrated into the miRNA basal segments inhibits Drosha processing, resulting in titratable control over gene silencing. The generality of this control strategy was shown for three aptamer–small molecule ligand pairs. The platform can be extended to the design of synthetic miRNAs clusters, cis-acting miRNAs and self-targeting miRNAs that act both in cis and trans, enabling fine-tuning of the regulatory strength and dynamics. The ability of our ligand-responsive miRNA platform to respond to user-defined inputs, undergo regulatory performance tuning and display scalable combinatorial control schemes will help advance applications in biological research and applied medicine

New RNA playgrounds : non-coding RNAs and RNA-binding proteins control cellular processes

Het eiwit Dead End noodzakelijk is voor het overleven van geslachtscellen. Het beschermt enkele genen tegen blokkades door microRNA__s. Dat stelt onderzoeker Martijn Kedde van het NKI-AVL in zijn proefschrift. Kedde promoveert donderdag 22 januari. MicroRNA__s, kleine stukjes RNA, blokkeren de ontwikkeling van geslachtscellen door enkele genen te remmen. Om te zorgen dat geslachtscellen zich toch kunnen ontwikkelen, is er het eiwit Dead End 1 (Dnd1). Het eiwit schermt het boodschapper-RNA van het erfelijk materiaal van de geslachtscel af zodat microRNA-430 er niet aan kan binden. In feite een doodlopende weg voor de microRNA__s. Ook heeft Kedde, onderzoeker in de groep van Reuven Agami, de rol van hTR in reactie op schade aan het DNA beschreven. hTR is onderdeel van het telomerasecomplex, een enzym dat er voor zorgt dat kankercellen ongebreideld kunnen delen. Het blijkt dat hTR de groei van kankercellen in de hand werkt doordat het schade aan het DNA negeert. De rode draad in het proefschrift van Kedde is non-coderend RNA. De functies van de verschillende non-coderende RNA__s die hij beschrijft, laten zien dat het speelveld (de __playground__) van niet-coderende RNA__s steeds maar weer groter blijkt dan eerder werd aangenomen.UBL - phd migration 201

Sexual health of people with disability and chronic illness

Dit proefschrift beschrijft vijf empirische studies, waarvan drie studies gericht zijn op het in kaart brengen van diverse aspecten van de seksuele gezondheid van mensen met een chronische ziekte of een lichamelijke beperking, en in het bijzonder van jonge vrouwen met borstkanker. Deze studies geven onder andere inzicht in de ernst en de aard van de seksuele problemen die mensen met een chronische ziekte of een lichamelijke beperking ervaren, alsook in de aandoenings- en behandelingsgerelateerde factoren die hiermee samenhangen. De overige twee studies richten zich op de effecten van een kortdurende psychoseksuele behandeling welke specifiek is ontwikkeld voor mensen met een chronische ziekte of lichamelijke beperking, en op het hulpzoekgedrag bij seksuele problemen en factoren die hiermee samenhangen. De belangrijkste bevindingen van dit proefschrift worden hieronder samengevat. Hulpzoekgedrag van mensen met een chronische ziekte of een lichamelijke beperking Mensen met een chronische ziekte of lichamelijke beperking hebben een veel grotere kans op problemen rondom seksualiteit en seksuele gezondheid dan de algemene bevolking. Diverse internationale studies hebben aangetoond dat zowel mannen als vrouwen met een lichamelijke aandoening, zoals bijvoorbeeld dwarslaesie, hersenbeschadiging, spierziekte, artritis, neurologische aandoening en spina bifida, minder tevreden zijn over hun seksueel leven en een lagere seksuele en lichamelijke zelfwaardering hebben in vergelijking met gezonde mannen en vrouwen. Dit impliceert dat deze groep ook vaker behoefte zal hebben aan hulp bij seksuele problemen. Alhoewel er diverse onderzoeken zijn uitgevoerd naar het hulpzoekgedrag bij seksuele problemen onder de algemene bevolking, is het onbekend of mensen met een ziekte of een beperking een geschikte hulpverlener weten te vinden

Pumilio directs deadenylation-associated translational repression of the cyclin-dependent kinase 1 activator RGC-32

Response gene to complement-32 (RGC-32) activates cyclin-dependent kinase 1, regulates the cell cycle and is deregulated in many human tumours. We previously showed that RGC-32 expression is upregulated by the cancer-associated Epstein-Barr virus (EBV) in latently infected B cells through the relief of translational repression. We now show that EBV infection of naïve primary B cells also induces RGC-32 protein translation. In EBV-immortalised cell lines, we found that RGC-32 depletion resulted in cell death, indicating a key role in B cell survival. Studying RGC-32 translational control in EBV-infected cells, we found that the RGC-32 3′untranslated region (3′UTR) mediates translational repression. Repression was dependent on a single Pumilio binding element (PBE) adjacent to the polyadenylation signal. Mutation of this PBE did not affect mRNA cleavage, but resulted in increased polyA tail length. Consistent with Pumilio-dependent recruitment of deadenylases, we found that depletion of Pumilio in EBV-infected cells increased RGC-32 protein expression and polyA tail length. The extent of Pumilio binding to the endogenous RGC-32 mRNA in EBV-infected cell lines also correlated with RGC-32 protein expression. Our data demonstrate the importance of RGC-32 for the survival of EBV-immortalised B cells and identify Pumilio as a key regulator of RGC-32 translation

Site identification in high-throughput RNA-protein interaction data

Motivation: Post-transcriptional and co-transcriptional regulation is a crucial link between genotype and phenotype. The central players are the RNA-binding proteins, and experimental technologies [such as cross-linking with immunoprecipitation-(CLIP-) and RIP-seq] for probing their activities have advanced rapidly over the course of the past decade. Statistically robust, flexible computational methods for binding site identification from high-throughput immunoprecipitation assays are largely lacking however.Results: We introduce a method for site identification which provides four key advantages over previous methods: (i) it can be applied on all variations of CLIP and RIP-seq technologies, (ii) it accurately models the underlying read-count distributions, (iii) it allows external covariates, such as transcript abundance (which we demonstrate is highly correlated with read count) to inform the site identification process and (iv) it allows for direct comparison of site usage across cell types or conditions. © The Author 2012. Published by Oxford University Press. All rights reserved

Evolution of the human-specific microRNA miR-941

MicroRNA-mediated gene regulation is important in many physiological processes. Here we explore the roles of a microRNA, miR-941, in human evolution. We find that miR-941 emerged de novo in the human lineage, between six and one million years ago, from an evolutionarily volatile tandem repeat sequence. Its copy-number remains polymorphic in humans and shows a trend for decreasing copy-number with migration out of Africa. Emergence of miR-941 was accompanied by accelerated loss of miR-941-binding sites, presumably to escape regulation. We further show that miR-941 is highly expressed in pluripotent cells, repressed upon differentiation and preferentially targets genes in hedgehog- and insulin-signalling pathways, thus suggesting roles in cellular differentiation. Human-specific effects of miR-941 regulation are detectable in the brain and affect genes involved in neurotransmitter signalling. Taken together, these results implicate miR-941 in human evolution, and provide an example of rapid regulatory evolution in the human linage

Verteporfin ameliorates fibrotic aspects of Dupuytren's disease nodular fibroblasts irrespective the activation state of the cells

Dupuytren's disease is a chronic, progressive fibroproliferative condition of the hand fascia which results in digital contraction. So far, treatments do not directly interfere with the (myo)fibroblasts that are responsible for the formation of the collagen-rich cords and its contraction. Here we investigated whether verteporfin (VP) is able to inhibit the activation and subsequent differentiation of DD nodular fibroblasts into myofibroblasts. Fibroblasts were isolated from nodules of 7 Dupuytren patients. Cells are treated (1) for 48 h with 5 ng/ml transforming growth factor β1 (TGF-β1) followed by 48 h with/without 250 nM VP in the absence of TGF-β1, or treated (2) for 48 h with TGF-β1 followed by 48 h with/without VP in the presence of TGF-β1. mRNA levels were measured by means of Real-Time PCR, and proteins were visualized by means of Western blotting and/or immunofluorescence. Quantitative data were statistically analyzed with GraphPad Prism using the paired t-test. We found that fibroblasts activated for 48 h with TGF-β1 show a decrease in mRNA levels of COL1A1, COL3A1, COL4A1, PLOD2, FN1EDA, CCN2 and SERPINE1 when exposed for another 48 h with VP, whereas no decrease is seen for ACTA2, YAP1, SMAD2 and SMAD3 mRNA levels. Cells exposed for an additional 48 h with TGF-β1, but now in the presence of VP, are not further activated anymore, whereas in the absence of VP the cells continue to differentiate into myofibroblasts. Collagen type I, fibronectin-extra domain A, α-smooth muscle actin, YAP1, Smad2 and Smad3 protein levels were attenuated by both VP treatments. We conclude that VP has strong anti-fibrotic properties: it is able to halt the differentiation of fibroblasts into myofibroblasts, and is also able to reverse the activation status of fibroblasts. The decreased protein levels of YAP1, Smad2 and Smad3 in the presence of VP explain in part the strong anti-fibrotic properties of VP. Verteporfin is clinically used as a photosensitizer for photodynamic therapy to eliminate abnormal blood vessels in the eye to attenuate macular degeneration. The antifibrotic properties of VP do not rely on photo-activation, as we used the molecule in its non-photoinduced state

HuR protein attenuates miRNA-mediated repression by promoting miRISC dissociation from the target RNA

The microRNA (miRNA)-mediated repression of protein synthesis in mammalian cells is a reversible process. Target mRNAs with regulatory AU-rich elements (AREs) in their 3′-untranslated regions (3′-UTR) can be relieved of miRNA repression under cellular stress in a process involving the embryonic lethal and altered vision family ARE-binding protein HuR. The HuR-mediated derepression occurred even when AREs were positioned at a considerable distance from the miRNA sites raising questions about the mechanism of HuR action. Here, we show that the relief of miRNA-mediated repression involving HuR can be recapitulated in different in vitro systems in the absence of stress, indicating that HuR alone is sufficient to relieve the miRNA repression upon binding to RNA ARE. Using in vitro assays with purified miRISC and recombinant HuR and its mutants, we show that HuR, likely by its property to oligomerize along RNA, leads to the dissociation of miRISC from target RNA even when miRISC and HuR binding sites are positioned at a distance. Further, we demonstrate that HuR association with AREs can also inhibit miRNA-mediated deadenylation of mRNA in the Krebs-2 ascites extract, in a manner likewise depending on the potential of HuR to oligomerize

AREsite: a database for the comprehensive investigation of AU-rich elements

AREsite is an online resource for the detailed investigation of AU-rich elements (ARE) in vertebrate mRNA 3′-untranslated regions (UTRs). AREs are one of the most prominent cis-acting regulatory elements found in 3′-UTRs of mRNAs. Various ARE-binding proteins that possess RNA stabilizing or destabilizing functions are recruited by sequence-specific motifs. Recent findings suggest an essential role of the structural mRNA context in which these sequence motifs are embedded. AREsite is the first database that allows to quantify the structuredness of ARE motif sites in terms of opening energies and accessibility probabilities. Moreover, we also provide a detailed phylogenetic analysis of ARE motifs and incorporate information about experimentally validated targets of the ARE-binding proteins TTP, HuR and Auf1. The database is publicly available at: http://rna.tbi.univie.ac.at/AREsite