Weiterentwicklung der Intramertechnologie zur Charakterisierung von Cytohesin-2 als neuen Effektor der MAP-Kaskade in HeLa-Zellen

Die Proteine der Klasse der kleinen Guaninnukleotid-Austauschfaktoren (GEF) katalysieren den Austausch von GDP gegen GTP an monomeren G-Proteinen der ARF-Familie und sind an der Regulation des Vesikeltransports beteiligt. Darüber hinaus übernehmen diese eine wichtige Rolle in einer Reihe unterschiedlicher zellulärer Prozesse wie der Reorganisation des Actincytoskeletts, der T-Zelladhäsion und der Zelldifferenzierung. Die Steuerung dieser Prozesse erfolgt u. a. durch die Interaktion von kleinen GEF mit αLβ2-Integrin. Darüber hinaus sind funktionale Wechselwirkungen mit anderen Proteinen bekannt, die allerdings nur unvollständig charakterisiert sind. Der Klasse der kleinen GEF werden vier Proteine zugeordnet, die als Cytohesin 1-4 bezeichnet werden. Diese weisen sowohl strukturell als auch funktionell eine große Homologie zueinander auf. Die zentrale Stellung und Komplexität der durch kleine Austauschfaktoren regulierten Prozesse unterstreicht die Bedeutung einer weiteren Charakterisierung und hebt die Notwendigkeit der Entwicklung neuer Werkzeuge zur biologischen Funktionsaufklärung hervor. In der vorliegenden Arbeit wurde ein RNA-Aptamer entwickelt, welches den Austauschfaktor Cytohesin-2 bindet und gegenüber Cytohesin-1 diskriminiert. Dieses durch in vitro Selektionstechnik erhaltene Aptamer bindet sein Zielprotein unter Beteiligung der Coiled-coil Domäne ohne dabei die Aktivität der Sec7- oder der PH-Domäne zu modulieren. Die Verwendung von Aptameren in Zellen (Intramere) ist bisher an den Einsatz eines Expressionssystems geknüpft. Diese Einschränkung konnte überwunden werden. So wurde das Anti-Cytohesin-2 Aptamer durch Lipofektion in HeLa-Zellen eingebracht. Eine zusätzliche Modifizierung oder Stabilisierung war nicht notwendig. Auf die zelluläre Transkriptionsmaschinerie oder die Verwendung eines Expressionssystems konnte verzichtet werden. Die funktionelle Charakterisierung von Cytohesin-2 mittels des spezifischen Intramers führte zur Aufklärung einer bis dato unbekannten Funktion als positiven Effektor der MAP-Kinasen Erk1/-2. So konnte die serumabhängige Expression von Genen unter der Kontrolle des „serum response elements“ (SRE) durch Inhibition von Cytohesin-2 blockiert werden. Das Bindungsepitop des Intramers legt dabei eine Beteiligung der Coiled-coil-Domäne von Cytohesin-2 an dieser Signaltransduktion nah. Durch Überexpression einer Sec7-defizienten Mutante von Cytohesin-2 konnte eine Beteiligung der Sec7-Domäne an der Modulation der MAP-Kinase Aktivität nachgewiesen werden. Eine regulatorische Funktion der Coiled-coil-Domäne bezüglich der Sec7-Aktivität erscheint somit als wahrscheinlich. Durch Verwendung von siRNA-Strategien, die entweder gegen Cytohesin-2 oder Cytohesin-1 gerichtet waren, konnte eine Beteiligung von Cytohesin-1 an der Regulation der Erk1/-2-Aktivität ausgeschlossen werden und unterstreicht zusätzlich die bereits durch das Intramer erzielten Resultate. Intramertechnologie als Werkzeug zur biologischen Funktionsaufklärung von Proteinen im Kontext einer lebenden Zelle weist ein hohes Potential auf und wird wohl in Zukunft eine größere Verbreitung in vielen Bereichen der Proteomforschung finden

Comparative profiling identifies C13orf3 as a component of the Ska complex required for mammalian cell division

Proliferation of mammalian cells requires the coordinated function of many proteins to accurately divide a cell into two daughter cells. Several RNAi screens have identified previously uncharacterised genes that are implicated in mammalian cell division. The molecular function for these genes needs to be investigated to place them into pathways. Phenotypic profiling is a useful method to assign putative functions to uncharacterised genes. Here, we show that the analysis of protein localisation is useful to refine a phenotypic profile. We show the utility of this approach by defining a function of the previously uncharacterised gene C13orf3 during cell division. C13orf3 localises to centrosomes, the mitotic spindle, kinetochores, spindle midzone, and the cleavage furrow during cell division and is specifically phosphorylated during mitosis. Furthermore, C13orf3 is required for centrosome integrity and anaphase onset. Depletion by RNAi leads to mitotic arrest in metaphase with an activation of the spindle assembly checkpoint and loss of sister chromatid cohesion. Proteomic analyses identify C13orf3 (Ska3) as a new component of the Ska complex and show a direct interaction with a regulatory subunit of the protein phosphatase PP2A. All together, these data identify C13orf3 as an important factor for metaphase to anaphase progression and highlight the potential of combined RNAi screening and protein localisation analyses

Concept of the Munich/Augsburg Consortium Precision in Mental Health for the German Center of Mental Health

The Federal Ministry of Education and Research (BMBF) issued a call for a new nationwide research network on mental disorders, the German Center of Mental Health (DZPG). The Munich/Augsburg consortium was selected to participate as one of six partner sites with its concept “Precision in Mental Health (PriMe): Understanding, predicting, and preventing chronicity.” PriMe bundles interdisciplinary research from the Ludwig-Maximilians-University (LMU), Technical University of Munich (TUM), University of Augsburg (UniA), Helmholtz Center Munich (HMGU), and Max Planck Institute of Psychiatry (MPIP) and has a focus on schizophrenia (SZ), bipolar disorder (BPD), and major depressive disorder (MDD). PriMe takes a longitudinal perspective on these three disorders from the at-risk stage to the first-episode, relapsing, and chronic stages. These disorders pose a major health burden because in up to 50% of patients they cause untreatable residual symptoms, which lead to early social and vocational disability, comorbidities, and excess mortality. PriMe aims at reducing mortality on different levels, e.g., reducing death by psychiatric and somatic comorbidities, and will approach this goal by addressing interdisciplinary and cross-sector approaches across the lifespan. PriMe aims to add a precision medicine framework to the DZPG that will propel deeper understanding, more accurate prediction, and personalized prevention to prevent disease chronicity and mortality across mental illnesses. This framework is structured along the translational chain and will be used by PriMe to innovate the preventive and therapeutic management of SZ, BPD, and MDD from rural to urban areas and from patients in early disease stages to patients with long-term disease courses. Research will build on platforms that include one on model systems, one on the identification and validation of predictive markers, one on the development of novel multimodal treatments, one on the regulation and strengthening of the uptake and dissemination of personalized treatments, and finally one on testing of the clinical effectiveness, utility, and scalability of such personalized treatments. In accordance with the translational chain, PriMe’s expertise includes the ability to integrate understanding of bio-behavioral processes based on innovative models, to translate this knowledge into clinical practice and to promote user participation in mental health research and care

A Genome-Scale DNA Repair RNAi Screen Identifies SPG48 as a Novel Gene Associated with Hereditary Spastic Paraplegia

We have identified a novel gene in a genome-wide, double-strand break DNA repair RNAi screen and show that is involved in the neurological disease hereditary spastic paraplegia

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs

An RNA interference phenotypic screen identifies a role for FGF signals in colon cancer progression

In tumor cells, stepwise oncogenic deregulation of signaling cascades induces alterations of cellular morphology and promotes the acquisition of malignant traits. Here, we identified a set of 21 genes, including FGF9, as determinants of tumor cell morphology by an RNA interference phenotypic screen in SW480 colon cancer cells. Using a panel of small molecular inhibitors, we subsequently established phenotypic effects, downstream signaling cascades, and associated gene expression signatures of FGF receptor signals. We found that inhibition of FGF signals induces epithelial cell adhesion and loss of motility in colon cancer cells. These effects are mediated via the mitogen-activated protein kinase (MAPK) and Rho GTPase cascades. In agreement with these findings, inhibition of the MEK1/2 or JNK cascades, but not of the PI3K-AKT signaling axis also induced epithelial cell morphology. Finally, we found that expression of FGF9 was strong in a subset of advanced colon cancers, and overexpression negatively correlated with patients' survival. Our functional and expression analyses suggest that FGF receptor signals can contribute to colon cancer progression

The long noncoding RNA <i>lncR492</i> inhibits neural differentiation of murine embryonic stem cells

<div><p>RNA interference (RNAi) screens have been shown to be valuable to study embryonic stem cell (ESC) self-renewal and they have been successfully applied to identify coding as well as noncoding genes required for maintaining pluripotency. Here, we used an RNAi library targeting >640 long noncoding RNAs (lncRNA) to probe for their role in early cell differentiation. Utilizing a Sox1-GFP ESC reporter cell line, we identified the lncRNA <i>lncR492</i> as lineage-specific inhibitor of neuroectodermal differentiation. Molecular characterization showed that <i>lncR492</i> interacts with the mRNA binding protein HuR and facilitates its inhibitory function by activation of Wnt signaling. Thus, lncRNAs modulate the fate decision of pluripotent stem cells.</p></div

HuR regulates ectodermal differentiation similar to <i>lncR492</i>.

<p>(A) Schematic overview over mass spectrometry after RNA pull-down.</p> <p>(B) Two-dimensional interaction blot for 1-450bp lncR492 mRNA fragment incubated with ESC total cell lysate identifies HuR (Elavl1) as possible binding partner.</p> <p>(C) Western blot against HuR after RNA pull down.</p> <p>(D) FACS analysis of % Sox1-GFP positive cells after esiRNA transfection and 4 days of differentiation. Knock-down of <i>lncR492</i> and HuR alone or in combination. Apc and Rad21 were targeted as controls. Data presents mean ± SD of 4 independent experiments.</p> <p>(E) FACS analysis of % Sox1-GFP+ cells after overexpression of HuR. Data presents mean ± SD of 4 independent experiments.</p> <p>(F) FACS analysis of % T-GFP+ cells after knock-down of HuR. Data presents mean ± SD of 4 independent experiments.</p> <p>(G) FACS analysis of % T-GFP+ cells after overexpression of HuR. Data presents mean ± SD of 4 independent experiments.</p> <p>* p<0.05; ** p<0.01; *** p<0.001; n.s.–not significant.</p

<i>LncR492</i> inhibits specifically neural differentiation.

<p>(A) Gene expression analysis by qRT-PCR in Sox1-GFP ESCs during 4 days of differentiation in N2B27. Data presents the mean ± SD of three independent experiments.</p> <p>(B) Immunofluorescent analysis of Sox1-GFP ESC after 72h of differentiation. Endogenous GFP (green), Tubb3 (red) and DAPI (blue) are shown. Scale bar 50 μm. Bar graph shows quantification of Tubb3 positive cells, which were normalized to the overall cell number.</p> <p>(C) Time course analysis of gene expression by qRT-PCR after <i>lncR492</i> knock-down in Sox1-GFP ESCs. Data presents mean ± SD of three independent experiments.</p> <p>(D) Transient overexpression of <i>lncR492</i> analysed by qRT-PCR in Sox1-GFP ESCs. Data presents the mean ± SD of three independent experiments. EV—empty vector.</p> <p>(E) FACS analysis of % Sox1-GFP positive cells after overexpression of <i>lncR492</i> and 4 days of differentiation. Data presents mean ± SD of 4 independent experiments.</p> <p>(F) FACS analysis of % T-GFP positive cells after overexpression of <i>lncR492</i> and 4 days of differentiation. Data presents mean ± SD of 4 independent experiments.</p> <p>(G) FACS analysis of % Foxa2-GFP positive cells after overexpression of <i>lncR492</i> and 4 days of differentiation. Data presents mean ± SD of 4 independent experiments.</p> <p>* p<0.05; ** p<0.01; *** p<0.001; n.s.–not significant.</p

Characterization of <i>lncR492</i>.

<p>(A) Schematic of the lncR492 locus. <i>lncR492</i> (accession no. AK016992) is located within the first intron of the protein-coding gene <i>Srrm4</i>.</p> <p>(B) Northern blot of <i>lncR494</i>. Increasing amounts of total RNA were loaded. Black arrow indicates <i>lncR492</i>-specific signal at ~1400 bp. A probe targeting <i>Gapdh</i> mRNA was used as loading control.</p> <p>(C) Analysis of polyadenylation. mRNA was transcribed into cDNA by using Oligo(dT) primer followed by PCR.</p> <p>(D) RT-PCR analysis of <i>lncR492</i> and <i>Gapdh</i> expression after the RNA extract was treated with a 5’-phosphate-dependent exonuclease, which results in a degradation of f.ex. ribosomal RNA (left panel).</p> <p>(E) RNA FISH of <i>lncR492</i> expression in undifferentiated ESC. The <i>lncR492</i>-specific signal (red) was reduced after <i>lncR492</i> knock-down. Scale bars are 10 μm.</p> <p>(F) Cellular fractionation in ESC was followed by RNA isolation and mRNA expression analysis by qRT-PCR.</p