TY - THES A1 - Kibe, Anuja T1 - Translational landscape and regulation of recoding in virus-infected cells T1 - Translationslandschaft und Regulierung der Rekodierung in virusinfizierten Zellen N2 - RNA viruses rely entirely on the host machinery for their protein synthesis and harbor non-canonical translation mechanisms, such as alternative initiation and programmed –1 ribosomal frameshifting (–1PRF), to suit their specific needs. On the other hand, host cells have developed a variety of defensive strategies to safeguard their translational apparatus and at times transiently shut down global translation. An infection can lead to substantial translational remodeling in cells and translational control is critical during antiviral response. Due to their sheer diversity, this control is likely unique to each RNA virus and the intricacies of post-transcriptional regulation are unclear in certain viral species. Here, we explored different aspects of translational regulation in virus-infected cells in detail. Using ribosome profiling, we extensively characterized the translational landscape in HIV-1 infected T cells, uncovering novel features of gene regulation in both host and virus. Additionally, we show that substantial pausing occurs prior to the frameshift site indicating complex regulatory mechanisms involving upstream viral RNA elements that can act as cis- regulators of frameshifting. We also characterized the mechanistic details of trans- modulation of frameshifting by host- and virus-encoded proteins. Host antiviral protein ZAP-S binds to the SARS-CoV-2 frameshift site and destabilizes the stimulatory structure, leading to frameshift inhibition. On the other hand, EMCV 2A protein stabilizes the viral frameshift site, thereby, activating EMCV frameshifting. While both proteins were shown to be antagonistic in their mechanism, they interact with the host translational machinery. Furthermore, we showed that frameshifting can be regulated not just by proteins, but also by small molecules. High-throughput screening of natural and synthetic compounds identified two potent frameshift inhibitors that also impeded viral replication, namely trichangion and compound 25. Together, this work largely enhances our understanding of gene regulation mechanisms in virus-infected cells and further validates the druggability of viral –1 PRF site. N2 - RNA-Viren sind bei der Proteinsynthese vollständig auf die Maschinerie des Wirts angewiesen und verfügen über nicht-kanonische Translationsmechanismen wie alternative Initiation und –1 programmiertes ribosomales Frameshifting (–1PRF), um ihre spezifischen Bedürfnisse zu erfüllen. Auf der anderen Seite haben die Wirtszellen eine Vielzahl von Abwehrstrategien entwickelt, um ihren Translationsapparat zu schützen und die globale Translation gegebenenfalls vorübergehend abzuschalten. Eine Infektion kann zu einer erheblichen Umgestaltung der Translation in den Zellen führen und die Kontrolle der Translation ist für die antivirale Reaktion von entscheidender Bedeutung. Aufgrund ihrer großen Vielfalt ist diese Kontrolle wahrscheinlich für jedes RNA-Virus einzigartig, und die Feinheiten der posttranskriptionellen Regulierung sind bei bestimmten Virusarten noch unklar. Hier haben wir verschiedene Aspekte der Translationsregulation in virusinfizierten Zellen im Detail untersucht. Mithilfe von Ribosomen-Profiling haben wir die Translationslandschaft in HIV-1-infizierten T-Zellen umfassend charakterisiert und dabei neue Merkmale der Genregulation sowohl im Wirt als auch im Virus aufgedeckt. Darüber hinaus konnten wir zeigen, dass Ribosomen vor der Frameshift-Stelle zu einem erheblichen Maße pausieren, was auf komplexe Regulationsmechanismen hinweist, an denen vorgelagerte virale RNA-Elemente beteiligt sind, die als cis-Regulatoren des Frameshifting wirken können. Darüber hinaus haben wir die mechanistischen Details der trans-Modulation des Frameshifting durch vom Wirt und vom Virus kodierte Proteine charakterisiert. Das antivirale Wirtsprotein ZAP-S bindet an die SARS-CoV-2 Frameshift-Stelle und destabilisiert die stimulierende Struktur, was zu einer Hemmung des Frameshifting führt. Auf der anderen Seite stabilisiert das EMCV-2A-Protein die virale Frameshift-Stelle und aktiviert dadurch das EMCV-Frameshifting. Obwohl sich beide Proteine in ihrem Mechanismus als antagonistisch erwiesen haben, interagieren sie mit der Translationsmaschinerie des Wirts. Darüber hinaus haben wir gezeigt, dass das Frameshifting nicht nur durch Proteine, sondern auch durch kleine Moleküle reguliert werden kann. Durch ein Hochdurchsatz-Screening natürlicher und synthetischer Verbindungen wurden zwei potente Frameshift-Inhibitoren identifiziert, die auch die virale Replikation behinderten, nämlich Trichangion und Compound 25. Zusammengenommen verbessert diese Arbeit unser Verständnis der Mechanismen der Genregulierung in virusinfizierten Zellen und bestätigt die Medikamentenfähigkeit der viralen -1 PRF-Seite. KW - Zelle KW - RNA virus KW - translation KW - ribosome profiling KW - programmed ribosomal frameshifting KW - RNS-Viren Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-310993 ER - TY - JOUR A1 - Chithelen, Janice A1 - Franke, Hannah A1 - Länder, Nora A1 - Grafen, Anika A1 - Schneider-Schaulies, Jürgen T1 - The sphingolipid inhibitors ceranib-2 and SKI-II reduce measles virus replication in primary human lymphocytes: effects on mTORC1 downstream signaling JF - Frontiers in Physiology N2 - The bioactive sphingolipids ceramide and sphingosine-1-phosphate (S1P) are involved in the regulation of cell homeostasis and activity ranging from apoptosis to proliferation. We recently described that the two compounds ceranib-2 (inhibiting acid ceramidase) and SKI-II [inhibiting the sphingosine kinases 1 and − 2 (SphK1/2)] reduce mTORC1 activity and measles virus (MV) replication in human primary peripheral blood lymphocytes (PBL) by about one log step. We now further investigated whether mTORC1 downstream signaling and viral protein expression may be affected by ceranib-2 and/or SKI-II. Western blot analyses showed that in uninfected cells the phosphorylation of the eukaryotic initiation factor 4E (eIF4E) was reduced by both inhibitors. Interestingly, MV infection led to an increase of rpS6 protein levels and phosphorylation of eIF4E. Treatment with both inhibitors reduced the rpS6 protein expression, and in addition, SKI-II reduced rpS6 phosphorylation. The phosphorylation of eIF4E was slightly reduced by both inhibitors. In addition, SKI-II led to reduced levels of IKK in MV-infected cells. Both inhibitors reduced the expression of viral proteins and the titers of newly synthesized MV by approximately one log step. As expected, SKI-II and rapamycin reduced also the virally encoded GFP expression; however, ceranib-2 astonishingly led to increased levels of GFP fluorescence. Our findings suggest that the inhibitors ceranib-2 and SKI-II act via differential mechanisms on MV replication. The observed effects on mTORC1 downstream signaling, predominantly the reduction of rpS6 levels by both inhibitors, may affect the translational capacity of the cells and contribute to the antiviral effect in human primary PBL. KW - acid ceramidase inhibitor ceranib-2 KW - sphingosine kinase inhibitor SKI-II KW - mTORC1 KW - translation KW - measles virus Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-265988 SN - 1664-042X VL - 13 ER - TY - JOUR A1 - Dugar, Gaurav A1 - Svensson, Sarah L. A1 - Bischler, Thorsten A1 - Waldchen, Sina A1 - Reinhardt, Richard A1 - Sauer, Markus A1 - Sharma, Cynthia M. T1 - The CsrA-FliW network controls polar localization of the dual-function flagellin mRNA in Campylobacter jejuni JF - Nature Communications N2 - The widespread CsrA/RsmA protein regulators repress translation by binding GGA motifs in bacterial mRNAs. CsrA activity is primarily controlled through sequestration by multiple small regulatory RNAs. Here we investigate CsrA activity control in the absence of antagonizing small RNAs by examining the CsrA regulon in the human pathogen Campylobacter jejuni. We use genome-wide co-immunoprecipitation combined with RNA sequencing to show that CsrA primarily binds flagellar mRNAs and identify the major flagellin mRNA (flaA) as the main CsrA target. The flaA mRNA is translationally repressed by CsrA, but it can also titrate CsrA activity. Together with the main C. jejuni CsrA antagonist, the FliW protein, flaA mRNA controls CsrA-mediated post-transcriptional regulation of other flagellar genes. RNA-FISH reveals that flaA mRNA is expressed and localized at the poles of elongating cells. Polar flaA mRNA localization is translation dependent and is post-transcriptionally regulated by the CsrA-FliW network. Overall, our results suggest a role for CsrA-FliW in spatiotemporal control of flagella assembly and localization of a dual-function mRNA. KW - bacterial genetics KW - cell signalling KW - translation KW - Campylobacter jejuni Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-173201 VL - 7 ER - TY - THES A1 - Pekárek, Lukáš T1 - Single-Molecule Approaches To Study Frameshifting Mechanisms T1 - Einzelmolekülansätze zur Untersuchung von Frameshifting-Mechanismen N2 - The RNAs of many viruses contain a frameshift stimulatory element (FSE) that grants access to an alternate reading frame via −1 programmed ribosomal frameshifting (PRF). This −1PRF is essential for effective viral replication. The −1PRF efficiency relies on the presence of conserved RNA elements within the FSE, such as a slippery sequence, spacer, and a downstream secondary structure – often a hairpin or a pseudoknot. The PRF efficiency is also affected by trans-acting factors such as proteins, miRNAs and metabolites. The interactions of these factors with the RNA and the translation machinery have not yet been completely understood. Traditional ensemble methods used previously to study these events focus on the whole population of molecular species. This results in innate averaging of the molecular behavior and a loss of heterogeneity information. Here, we first established the experimental workflow to study the RNA structures and the effect of potential trans-acting factors using single-molecule force spectroscopy technique, optical tweezers. Additionally, to streamline the data analysis, we developed an algorithm for automatized data processing. Next, we harnessed this knowledge to study viral RNA elements responsible for stimulation of PRF and how the presence of trans-acting factors affects the RNA behavior. We further complemented these single-molecule structural data with ensemble functional assays to gain a complex view on the dynamics behind the programmed ribosomal frameshifting. Specifically, two different viral RNA elements have been studied in the presented work. First, the dynamics of SARS-CoV-2 FSE and the role of extended sequences have been explored. Then, the mode of action of the host-encoded trans-acting factor ZAP-S inhibition of SARS-CoV-2 PRF has been examined. Finally, the mechanism of the trans-acting viral factor induced PRF in Encephalomyocarditis virus (EMCV) has been uncovered. N2 - Die RNAs vieler Viren enthalten ein Lese-Rasterverschiebung-stimulierendes Element (FSE), das über die −1 programmierte ribosomale Rasterverschiebung (PRF) Zugriff auf einen alternativen Leserahmen gewährt. Dieser −1PRF ist für eine effektive Virusreplikation unerlässlich. Die −1PRF-Effizienz beruht auf dem Vorhandensein konservierter RNA-Elemente innerhalb des FSE, wie z.B. einer Slippery-Sequenz, einem Platzhalter und einer nachgelagerten Sekundärstruktur – oft eine Haarnadel oder ein Pseudoknoten. Die −1PRF-Effizienz wird auch durch trans-aktive Faktoren wie Proteine, miRNAs und Metaboliten beeinflusst. Die Wechselwirkungen dieser Faktoren mit der RNA und der Translationsmaschinerie sind noch nicht vollständig verstanden. Traditionelle Ensemble-Methoden, die früher zur Untersuchung dieser Ereignisse verwendet wurden, konzentrieren sich auf die gesamte Population molekularer Spezies. Dies führt zu einer inhärenten Durchschnittsbildung des molekularen Verhaltens und einem Verlust von Heterogenitätsinformationen. Hier haben wir zunächst den experimentellen Arbeitsablauf zur Untersuchung der RNA-Strukturen und der Wirkung potenzieller trans-aktiver Faktoren mithilfe der Einzelmolekül-Kraftspektroskopietechnik Optischer Pinzetten etabliert. Um die Datenanalyse zu optimieren, haben wir außerdem einen Algorithmus zur automatisierten Datenverarbeitung entwickelt. Als nächstes nutzten wir dieses Wissen, um virale RNA-Elemente zu untersuchen, die für die Stimulierung von −1PRF verantwortlich sind, und wie sich das Vorhandensein trans-aktiver Faktoren auf das Verhalten der RNA auswirkt. Wir haben diese Einzelmolekülstrukturdaten weiter durch Ensemble-Funktionsassays ergänzt, um einen komplexen Überblick über die Dynamik hinter der programmierten ribosomalen Rasterverschiebung zu erhalten. Konkret wurden in der vorgestellten Arbeit zwei verschiedene virale RNA-Elemente untersucht. Zunächst wurden die Dynamik des SARS-CoV-2-FSE und die Rolle erweiterter Sequenzen untersucht. Anschließend wurde die hemmende Wirkungsweise des vom Wirt kodierten trans-wirkenden Faktors ZAP-S auf SARS-CoV-2-PRF untersucht. Schließlich wurde der Mechanismus der, durch den trans-aktiven Virusfaktor induzierten PRF beim Enzephalomyokarditis-Virus (EMCV), entschlüsselt KW - translation KW - infection KW - frameshifting Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-346112 ER - TY - JOUR A1 - Zukher, Inna A1 - Novikova, Maria A1 - Tikhonov, Anton A1 - Nesterchuk, Mikhail V. A1 - Osterman, Ilya A. A1 - Djordjevic, Marko A1 - Sergiev, Petr V. A1 - Sharma, Cynthia M. A1 - Severinov, Konstantin T1 - Ribosome-controlled transcription termination is essential for the production of antibiotic microcin C JF - Nucleic Acids Research N2 - Microcin C (McC) is a peptide-nucleotide antibiotic produced by Escherichia coli cells harboring a plasmid-borne operon mccABCDE. The heptapeptide MccA is converted into McC by adenylation catalyzed by the MccB enzyme. Since MccA is a substrate for MccB, a mechanism that regulates the MccA/MccB ratio likely exists. Here, we show that transcription from a promoter located upstream of mccA directs the synthesis of two transcripts: a short highly abundant transcript containing the mccA ORF and a longer minor transcript containing mccA and downstream ORFs. The short transcript is generated when RNA polymerase terminates transcription at an intrinsic terminator located in the intergenic region between the mccA and mccB genes. The function of this terminator is strongly attenuated by upstream mcc sequences. Attenuation is relieved and transcription termination is induced when ribosome binds to the mccA ORF. Ribosome binding also makes the mccA RNA exceptionally stable. Together, these two effects-ribosome induced transcription termination and stabilization of the message-account for very high abundance of the mccA transcript that is essential for McC production. The general scheme appears to be evolutionary conserved as ribosome-induced transcription termination also occurs in a homologous operon from Helicobacter pylori. KW - escherichia coli KW - messenger-RNA decay KW - translation KW - expression KW - synthetase KW - enterobacteria KW - inhibitors KW - maturation KW - target KW - stability Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-114839 SN - 0305-1048 VL - 42 IS - 19 ER - TY - JOUR A1 - Preising, Christina A1 - Schneider, Reinhard A1 - Bucher, Michael A1 - Gekle, Michael A1 - Sauvant, Christoph T1 - Regulation of expression of renal organic anion transporters OAT1 and OAT3 in a model of ischemia/reperfusion injury JF - Cellular Physiology and Biochemistry N2 - Background: Recently, we gained evidence that impairment of rOat1 and rOat3 expression induced by ischemic acute kidney injury (AKI) is mediated by COX metabolites and this suppression might be critically involved in renal damage. Methods: (i) Basolateral organic anion uptake into proximal tubular cells after model ischemia and reperfusion (I/R) was investigated by fluorescein uptake. The putative promoter sequences from hOAT1 (SLC22A6) and hOAT3 (SCL22A8) were cloned into a reporter plasmid, transfected into HEK cells and (ii) transcriptional activity was determined after model ischemia and reperfusion as a SEAP reporter gen assay. Inhibitors or antagonists were applied with the beginning of reperfusion. Results: By using inhibitors of PKA (H89) and PLC (U73122), antagonists of E prostanoid receptor type 2 (AH6809) and type 4 (L161,982), we gained evidence that I/R induced down regulation of organic anion transport is mediated by COX1 metabolites via E prostanoid receptor type 4. The latter signaling was confirmed by application of butaprost (EP2 agonist) or TCS2510 (EP4 agonist) to control cells. In brief, the latter signaling was verified for the transcriptional activity in the reporter gen assay established. Therein, selective inhibitors for COX1 (SC58125) and COX2 (SC560) were also applied. Conclusion: Our data show (a) that COX1 metabolites are involved in the regulation of renal organic anion transport(ers) after I/R via the EP4 receptor and (b) that this is due to transcriptional regulation of the respective transporters. As the promoter sequences cloned were of human origin and expressed in a human renal epithelial cell line we (c) hypothesize that the regulatory mechanisms described after I/R is meaningful for humans as well. KW - opossum kidney cells KW - prostaglandin e2 KW - reperfusion KW - transport experiments KW - translation KW - reporter gen assay KW - cloning of putative human promoter sequence KW - regulation of expression KW - OAT1 KW - OAT3 KW - OK cells KW - ischemic acute kidney injury model KW - HEK cells KW - ischemia KW - down regulation KW - nitric oxide KW - cellular physiology KW - cortical OAT1 KW - blood flow Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-144504 VL - 37 IS - 1 ER - TY - THES A1 - Schäffler, Katrin M. T1 - Regulation der eukaryotischen Translation durch RNA-bindende Faktoren: Strukturelle und funktionelle Charakterisierung des La-verwandten Proteins 4B (LARP4B) T1 - Regulation of the eukaryotic translation by RNA-binding factors: structural and functional characterization of the La-related protein 4B (LARP4B) N2 - In Zellen liegen RNAs in Form von Ribonukleoprotein-Komplexen (RNP) vor, wobei das Zusammenwirken von RNA und Proteinen die Funktionen der einzelnen RNPs definiert. RNA-bindenden Proteinen kommt demnach eine zentrale Bedeutung beim Verständnis des RNA-Metabolismus zu. Zu dieser Proteingruppe zählen auch die La-verwandten Proteine (engl. La-related proteins, LARPs), welche eine evolutionär konservierte Familie von Faktoren bilden und durch eine putative RNA-bindende Domäne, dem La Modul, charakterisiert sind. Bereits für zwei Vertreter dieser Proteinklasse (LARP3 und LARP7) konnte eine über das La Modul vermittelte spezifische Interaktion mit uridylreichen RNA-Sequenzen gezeigt werden. Ziel dieser Arbeit war es, einen Vertreter der LARP-Familie, das sogenannte LARP4B, sowohl biochemisch als auch strukturell zu untersuchen und es somit einem zellulären Prozess zuzuordnen. Zellbiologische Studien zeigten zunächst, dass LARP4B unter normalen Wachstumsbedingungen eine homogene zytoplasmatische Verteilung aufweist. Unter Stressbedingungen akkumuliert LARP4B hingegen in diskreten subzellulären Domänen, den sogenannten Stress Granules (SGs). Obwohl SGs bislang noch wenig funktionell untersucht sind, wird davon ausgegangen, dass sie der reversiblen Speicherung von mRNA-gebundenen Translationsfaktoren dienen. Durch affinitätschromatographische Strategien ließen sich spezifische Interaktionspartner von LARP4B identifizieren. Als direkte Bindungspartner wurden das zytoplasmatische Poly (A) bindende Protein 1 (PABPC1) und der Rezeptor für aktivierte C Kinase 1 (RACK1) gefunden. Darüber hinaus zeigten Sedimentationsanalysen, dass LARP4B nahezu quantitativ mit Ribosomen und Polyribosomen assoziiert vorliegt. Diese Studie identifizierte daher LARP4B als ein Protein, das mit Schlüsselfaktoren der eukaryotischen Translation wechselwirkt. In Übereinstimmung mit diesen Befunden reduziert ein RNAi-induzierter Mangel des Proteins die Translationsrate drastisch, während die Überexpression von LARP4B in vivo zu einer Stimulation der Proteinbiosynthese führt. Da dieser stimulatorische Einfluss bei einer Vielzahl unterschiedlicher mRNA-Spezies detektiert werden konnte, kann LARP4B als genereller, positiver Translationsfaktor angesehen werden. Interessanterweise wurden in Studien, die zeitgleich für das verwandte LARP1 durchgeführt wurden, vergleichbare zelluläre Interaktionen wie für LARP4B beschrieben. Um zu klären, ob beide LARPs Orthologe darstellen und funktionelle Redundanz zeigen, wurde in der vorgelegten Arbeit ein Vergleich von LARP4B mit LARP1 durchgeführt. Unabhängige in vivo Studien und Sedimentationsanalysen zeigten deutlich, dass beide Proteine im mRNA-Metabolismus agieren, jedoch in diesem unterschiedliche Phasen der eukaryotischen Proteinbiosynthese beeinflussen. N2 - The cooperation of RNA with different classes of proteins in so called ribonucleoprotein complexes (RNPs) is essential for the function of these RNPs. Therefore, RNA-binding proteins play a crucial role to understand the complex mechanisms of RNA-metabolism. One family of such proteins comprise the La-related proteins (LARPs). These evolutionary conserved factors are characterized by a putative RNA-binding domain, named the La module. For two of these factors (LARP3 and LARP7) a specific interaction with RNA containing uridine-rich sequence elements mediated via their La module could be described. The present work describes the biochemical and structural characterization of LARP4B, a thus far uncharacterized member of the LARP family. Immunofluorescence analyses identified LARP4B as a cytosolic protein that accumulates upon arsenite treatment in cellular stress granules (SGs). While still not sufficiently determined, these domains are believed to serve as storage pools for stalled, mRNA-bound translation initiation complexes formed upon polyribosome disassembly. Biochemical experiments further uncovered an interaction of LARP4B with the Poly(A) binding protein cytosolic 1 (PABPC1) and the receptor for activated C Kinase 1 (RACK1). Moreover, under physiological conditions, LARP4B co-sediments almost quantitatively with polysomes in cellular extracts, suggesting a role in translation. In agreement with this notion, knockdown of LARP4B by RNA-interference impaired translation of cellular mRNAs whereas over-expression stimulated protein synthesis. As this stimulatory effect could be detected for a wide range of different mRNA-species, LARP4B hence represents a general stimulator of translation. Interestingly, parallel studies uncovered comparable cellular interactions for another LARP family member (LARP1). To test whether LARP4B and LARP1 represent orthologs possessing redundant function, these two factors have been compared in this work using several independent in vivo and in vitro studies. These data clearly showed that both proteins positively influence RNA-metabolism but influence different phases of protein biosynthesis. KW - Translation KW - Eukaryoten KW - Ribonucleoproteine KW - Regulation KW - LARP4B KW - La Protein KW - PABPC1 KW - RACK1 KW - translation KW - LARP4B KW - La protein KW - PABPC1 KW - RACK1 Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-69809 ER - TY - JOUR A1 - Minnerup, Jens A1 - Sutherland, Brad A. A1 - Buchan, Alastair M. A1 - Kleinschnitz, Christoph T1 - Neuroprotection for Stroke: Current Status and Future Perspectives JF - International Journal of Molecular Science N2 - Neuroprotection aims to prevent salvageable neurons from dying. Despite showing efficacy in experimental stroke studies, the concept of neuroprotection has failed in clinical trials. Reasons for the translational difficulties include a lack of methodological agreement between preclinical and clinical studies and the heterogeneity of stroke in humans compared to homogeneous strokes in animal models. Even when the international recommendations for preclinical stroke research, the Stroke Academic Industry Roundtable (STAIR) criteria, were followed, we have still seen limited success in the clinic, examples being NXY-059 and haematopoietic growth factors which fulfilled nearly all the STAIR criteria. However, there are a number of neuroprotective treatments under investigation in clinical trials such as hypothermia and ebselen. Moreover, promising neuroprotective treatments based on a deeper understanding of the complex pathophysiology of ischemic stroke such as inhibitors of NADPH oxidases and PSD-95 are currently evaluated in preclinical studies. Further concepts to improve translation include the investigation of neuroprotectants in multicenter preclinical Phase III-type studies, improved animal models, and close alignment between clinical trial and preclinical methodologies. Future successful translation will require both new concepts for preclinical testing and innovative approaches based on mechanistic insights into the ischemic cascade. KW - free radical scavenger KW - ischemic cascade KW - acute ischemic stroke KW - trial KW - focal cerebral-ischemia KW - interleukin-1 receptor antagonist KW - colony-stimulating factor KW - tissue-plasminogen activator KW - traumatic brain injury KW - placebo-controlled KW - alias pilot trial KW - damage cool aid KW - neuroprotection KW - ischemic stroke KW - translation KW - STAIR Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-134730 VL - 13 IS - 9 ER - TY - JOUR A1 - Whisnant, Adam W. A1 - Jürges, Christopher S. A1 - Hennig, Thomas A1 - Wyler, Emanuel A1 - Prusty, Bhupesh A1 - Rutkowski, Andrzej J. A1 - L'hernault, Anne A1 - Djakovic, Lara A1 - Göbel, Margarete A1 - Döring, Kristina A1 - Menegatti, Jennifer A1 - Antrobus, Robin A1 - Matheson, Nicholas J. A1 - Künzig, Florian W. H. A1 - Mastrobuoni, Guido A1 - Bielow, Chris A1 - Kempa, Stefan A1 - Liang, Chunguang A1 - Dandekar, Thomas A1 - Zimmer, Ralf A1 - Landthaler, Markus A1 - Grässer, Friedrich A1 - Lehner, Paul J. A1 - Friedel, Caroline C. A1 - Erhard, Florian A1 - Dölken, Lars T1 - Integrative functional genomics decodes herpes simplex virus 1 JF - Nature Communications N2 - The predicted 80 open reading frames (ORFs) of herpes simplex virus 1 (HSV-1) have been intensively studied for decades. Here, we unravel the complete viral transcriptome and translatome during lytic infection with base-pair resolution by computational integration of multi-omics data. We identify a total of 201 transcripts and 284 ORFs including all known and 46 novel large ORFs. This includes a so far unknown ORF in the locus deleted in the FDA-approved oncolytic virus Imlygic. Multiple transcript isoforms expressed from individual gene loci explain translation of the vast majority of ORFs as well as N-terminal extensions (NTEs) and truncations. We show that NTEs with non-canonical start codons govern the subcellular protein localization and packaging of key viral regulators and structural proteins. We extend the current nomenclature to include all viral gene products and provide a genome browser that visualizes all the obtained data from whole genome to single-nucleotide resolution. Here, using computational integration of multi-omics data, the authors provide a detailed transcriptome and translatome of herpes simplex virus 1 (HSV-1), including previously unidentified ORFs and N-terminal extensions. The study also provides a HSV-1 genome browser and should be a valuable resource for further research. KW - infected-cell protein KW - messenger RNA KW - binding protein KW - type 1 KW - identification KW - ICP27 KW - translation KW - expression KW - sequence KW - domain Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-229884 VL - 11 ER - TY - THES A1 - Surrey, Verena T1 - Identification of affected cellular targets, mechanisms and signaling pathways in a mouse model for spinal muscular atrophy with respiratory distress type 1 (SMARD1) T1 - Identifizierung betroffener zellulärer Zielmoleküle, Mechanismen und Signalwege in einem Maus-Modell für spinale Muskelatrophie mit Ateminsuffizienz Typ 1 (SMARD1) N2 - Spinal muscular atrophy with respiratory distress type 1 (SMARD1) is a fatal monogenic motoneuron disease in children with unknown etiology caused by mutations in the immunoglobulin μ-binding protein 2 (IGHMBP2) gene coding for DNA/RNA ATPase/helicase. Despite detailed knowledge of the underlying genetic changes, the cellular mechanisms leading to this disease are not well understood. In the Nmd2J ("neuromuscular disorder") mouse, the mouse model for the juvenile form of SMARD1 patients, in which similar pathological features as diaphragmatic paralysis and skeletal muscle atrophy are observed. Ex vivo studies in Nmd2J mice showed that loss of the motor axon precedes atrophy of the gastrocnemius muscle and does not correlate with neurotransmission defects in the motor endplate. The already described independent myogenic anomalies in the diaphragm and heart of the Nmd2J mouse raised the question whether spinal motoneuron degeneration develops cell autonomously. Ighmbp2 is predominantly localized in the cytoplasm and seems to bind to ribosomes and polysomes, suggesting a role in mRNA metabolism. In this Ph.D. thesis, morphological and functional analyses of isolated Ighmbp2-deficient (Ighmbp2-def.) motoneurons were performed to answer the question whether the SMARD1 phenotype results from dysregulation of protein biosynthesis. Ighmbp2-deficient motoneurons show only negligible morphological alterations with respect to a slight increase in axonal branches. This observation is consistent with only minor changes of transcriptome based on RNA sequencing data from Ighmbp2-deficient motoneurons. Only the mRNA of fibroblast growth factor receptor 1 (Fgfr1) showed significant up-regulation in Ighmbp2-deficient motoneurons. Furthermore, no global aberrations at the translational level could be detected using pulsed SILAC (Stable Isotope Labeling by Amino acids in cell culture), AHA (L-azidohomoalanine) labeling and SUnSET (SUrface SEnsing of Translation) methods. However, a reduced β-actin protein level was observed at the growth cones of Ighmbp2-deficient motoneurons, which was accompanied with a reduced level of Imp1 protein, a known β-actin mRNA interactor. Live-cell imaging studies using fluorescence recovery after photobleaching (FRAP) showed translational down-regulation of eGFPmyr-β-actin 3'UTR mRNA in the growth cones and the cell bodies, although the amount of β-actin mRNA and the total protein amount in Ighmbp2-deficient motoneurons showed no aberrations. This compartment-specific reduction of β-actin protein occurred independently of a non-existent direct IGHMBPF2 binding to β-actin mRNA. Fgfr1, which was upregulated on the RNA level, did not show an increased protein amount in Ighmbp2-deficient motoneurons, whereas a reduced amount could be detected. Interestingly, a correlation could be found between the reduced amount of the Imp1 protein and the increased Fgfr1 mRNA, since the IMP1 protein binds the FGFR1 mRNA and thus could influence the transport and translation of FGFR1 mRNA. In summary, all data suggest that Ighmbp2 deficiency leads to a local but modest disturbance of protein biosynthesis, which might contribute to the motoneuron defects of SMARD1. N2 - Die spinale Muskelatrophie mit Atemnot Typ 1 (SMARD1) ist eine tödliche, monogene Motoneuron-Erkrankung bei Kindern mit unbekannter Ätiologie. SMARD1 wird durch Mutationen im Immunoglobulin µ-bindenden Protein 2 (IGHMBP2)-Gen verursacht, welches für eine DNA/RNA ATPase/Helikase kodiert. Trotz detaillierter Kenntnisse über die zugrunde liegenden genetischen Veränderungen sind die zellulären Mechanismen, die zu dieser Krankheit führen, nicht gut verstanden. In der Nmd2J („neuromuscular disorder“) Maus, dem Mausmodell für die juvenile Form von SMARD1-Patienten, werden ähnliche pathologische Merkmale wie Diaphragma-Lähmung und Skelettmuskelatrophie beobachtet. Ex vivo-Studien an Nmd2J-Mäusen zeigten, dass der Verlust des motorischen Axons einer Atrophie des Gastrocnemius-Muskels vorausgeht und nicht mit Neurotransmissionsfehlern an der motorischen Endplatte korreliert. Die bereits beschriebenen, unabhängig auftretenden myogenen Anomalien in Zwerchfell und Herz der Nmd2J-Maus führten zu der Frage, ob sich die spinale Motoneuron-Degeneration zellautonom entwickelt. Ighmbp2 ist prädominant im Zytoplasma lokalisiert und scheint an Ribosomen und Polysomen zu binden, was auf eine Rolle im mRNA-Stoffwechsel hindeutet. In dieser Doktorarbeit wurden morphologische und funktionelle Analysen von isolierten Ighmbp2-defizienten (Ighmbp2-def.) Motoneuronen durchgeführt, um die Frage zu beantworten, ob der SMARD1-Phänotyp aus der Deregulierung der Proteinbiosynthese resultiert. Ighmbp2-defiziente Motoneuronen weisen nur geringfügige morphologische Unterschiede hinsichtlich einer leichten Zunahme der axonalen Verzweigungen auf. Diese Beobachtung steht im Einklang mit nur geringen Veränderungen im Transkriptom basierend auf den RNA-Sequenzierungs-Daten in Ighmbp2-defizienten Motoneuronen. Ausschließlich die mRNA des Fibroblasten-Wachstumsfaktor-Rezeptor 1 (Fgfr1) zeigte eine signifikante Hoch-Regulation in Ighmbp2-defizienten Motoneuronen. Des Weiteren konnten keine globalen Aberrationen auf der translationalen Ebene mit Hilfe der gepulsten SILAC (Stable Isotope Labeling by Amino acids in der Zellkultur), AHA (L-Azidohomoalanin)-Markierung und der SUnSET (SUrface SEnsing of Translation) Methoden ermittelt werden. Jedoch konnte eine verringerte β-actin Proteinmenge an den Wachstumskegeln von Ighmbp2-defizienten Motoneuronen beobachtet werden, die von einer Reduktion an Imp1 Protein, einem bekannten β-actin mRNA Interaktor, begleitet wurde. Lebendzell-Bildgebungsstudien mittels Fluoreszenz-Recovery after Photobleaching (FRAP) Untersuchung zeigten eine translatorische Herunter-Regulation der eGFPmyr-β-actin 3‘UTR mRNA in Wachstumskegeln und Zellkörpern, obwohl die Menge an β-actin mRNA und die Gesamt-Proteinmenge in Ighmbp2-defizienten Motoneuronen im Vergleich zu wildtypischen Motoneuronen unverändert war. Diese Kompartiment-spezifische Reduktion von β-actin Protein trat unabhängig von einer nicht vorhandenen direkten IGHMBP2-Bindung an die β-actin mRNA auf. Der auf RNA Ebene hochregulierte Fgfr1 zeigte hingegen keine erhöhte, aber eine verringerte Proteinmenge in Ighmbp2-defizienten Motoneuronen. Interessanterweise konnte ein Zusammenhang zwischen der reduzierten Menge des Imp1 Proteins und der erhöhten FGFR1 mRNA gezogen werden. Da das IMP1 Protein neben der β-actin mRNA ebenfalls die FGFR1 mRNA bindet, könnte es so den Transport und die Translation beeinflussen. Alle Daten deuten zusammenfassend daraufhin, dass Ighmbp2-Mangel zu einer lokalen und geringen Störung der Proteinbiosynthese führt, die aber durchaus zu den beobachteten Motoneuron-Defekten in SMARD1 beitragen könnte. KW - Spinale Muskelatrophie KW - Ighmbp2 KW - Maus KW - RNA helicase KW - IMP1/ZBP1 KW - SMARD1 KW - motoneuron KW - translation KW - Signalkette KW - Molekularbiologie KW - Atmungsinsuffizienz Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-176386 ER - TY - JOUR A1 - Wencker, Freya D. R A1 - Marincola, Gabriella A1 - Schoenfelder, Sonja M. K. A1 - Maaß, Sandra A1 - Becher, Dörte A1 - Ziebuhr, Wilma T1 - Another layer of complexity in Staphylococcus aureus methionine biosynthesis control: unusual RNase III-driven T-box riboswitch cleavage determines met operon mRNA stability and decay JF - Nucleic Acids Research N2 - In Staphylococcus aureus, de novo methionine biosynthesis is regulated by a unique hierarchical pathway involving stringent-response controlled CodY repression in combination with a T-box riboswitch and RNA decay. The T-box riboswitch residing in the 5′ untranslated region (met leader RNA) of the S. aureus metICFE-mdh operon controls downstream gene transcription upon interaction with uncharged methionyl-tRNA. met leader and metICFE-mdh (m)RNAs undergo RNase-mediated degradation in a process whose molecular details are poorly understood. Here we determined the secondary structure of the met leader RNA and found the element to harbor, beyond other conserved T-box riboswitch structural features, a terminator helix which is target for RNase III endoribonucleolytic cleavage. As the terminator is a thermodynamically highly stable structure, it also forms posttranscriptionally in met leader/ metICFE-mdh read-through transcripts. Cleavage by RNase III releases the met leader from metICFE-mdh mRNA and initiates RNase J-mediated degradation of the mRNA from the 5′-end. Of note, metICFE-mdh mRNA stability varies over the length of the transcript with a longer lifespan towards the 3′-end. The obtained data suggest that coordinated RNA decay represents another checkpoint in a complex regulatory network that adjusts costly methionine biosynthesis to current metabolic requirements. KW - allelic replacement KW - expression KW - translation KW - mechanism KW - acid KW - endoribonuclease KW - antitermination KW - transcription KW - proteins KW - geometry Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-259029 VL - 49 IS - 4 ER - TY - JOUR A1 - Libre, Camille A1 - Seissler, Tanja A1 - Guerrero, Santiago A1 - Batisse, Julien A1 - Verriez, Cédric A1 - Stupfler, Benjamin A1 - Gilmer, Orian A1 - Cabrera-Rodriguez, Romina A1 - Weber, Melanie M. A1 - Valenzuela-Fernandez, Agustin A1 - Cimarelli, Andrea A1 - Etienne, Lucie A1 - Marquet, Roland A1 - Paillart, Jean-Christophe T1 - A conserved uORF regulates APOBEC3G translation and is targeted by HIV-1 Vif protein to repress the antiviral factor JF - Biomedicines N2 - The HIV-1 Vif protein is essential for viral fitness and pathogenicity. Vif decreases expression of cellular restriction factors APOBEC3G (A3G), A3F, A3D and A3H, which inhibit HIV-1 replication by inducing hypermutation during reverse transcription. Vif counteracts A3G at several levels (transcription, translation, and protein degradation) that altogether reduce the levels of A3G in cells and prevent its incorporation into viral particles. How Vif affects A3G translation remains unclear. Here, we uncovered the importance of a short conserved uORF (upstream ORF) located within two critical stem-loop structures of the 5′ untranslated region (5′-UTR) of A3G mRNA for this process. A3G translation occurs through a combination of leaky scanning and translation re-initiation and the presence of an intact uORF decreases the extent of global A3G translation under normal conditions. Interestingly, the uORF is also absolutely required for Vif-mediated translation inhibition and redirection of A3G mRNA into stress granules. Overall, we discovered that A3G translation is regulated by a small uORF conserved in the human population and that Vif uses this specific feature to repress its translation. KW - HIV-1 KW - APOBEC3G KW - Vif KW - mRNA KW - translation KW - uORF Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-252147 SN - 2227-9059 VL - 10 IS - 1 ER -