TY  - JOUR
A1  - Schlecht, Anja
A1  - Wolf, Julian
A1  - Boneva, Stefaniya
A1  - Prinz, Gabriele
A1  - Braunger, Barbara M.
A1  - Wieghofer, Peter
A1  - Agostini, Hansjürgen
A1  - Schlunck, Günther
A1  - Lange, Clemens
T1  - Transcriptional and distributional profiling of microglia in retinal angiomatous proliferation
JF  - International Journal of Molecular Sciences
N2  - Macular neovascularization type 3, formerly known as retinal angiomatous proliferation (RAP), is a hallmark of age-related macular degeneration and is associated with an accumulation of myeloid cells, such as microglia (MG) and infiltrating blood-derived macrophages (MAC). However, the contribution of MG and MAC to the myeloid cell pool at RAP sites and their exact functions remain unknown. In this study, we combined a microglia-specific reporter mouse line with a mouse model for RAP to identify the contribution of MG and MAC to myeloid cell accumulation at RAP and determined the transcriptional profile of MG using RNA sequencing. We found that MG are the most abundant myeloid cell population around RAP, whereas MAC are rarely, if ever, associated with late stages of RAP. RNA sequencing of RAP-associated MG showed that differentially expressed genes mainly contribute to immune-associated processes, including chemotaxis and migration in early RAP and proliferative capacity in late RAP, which was confirmed by immunohistochemistry. Interestingly, MG upregulated only a few angiomodulatory factors, suggesting a rather low angiogenic potential. In summary, we showed that MG are the dominant myeloid cell population at RAP sites. Moreover, MG significantly altered their transcriptional profile during RAP formation, activating immune-associated processes and exhibiting enhanced proliferation, however, without showing substantial upregulation of angiomodulatory factors.
KW  - AMD
KW  - Mactel 2
KW  - macular neovascularization
KW  - MNV type 3
KW  - retinal angiomatous proliferation
KW  - RAP
KW  - microglia
KW  - RNA sequencing
KW  - Cx3cr1
KW  - CreERT2
Y1  - 2022
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-284072
SN  - 1422-0067
VL  - 23
IS  - 7
ER  - 
TY  - JOUR
A1  - Esken, Jens
A1  - Goris, Tobias
A1  - Gadkari, Jennifer
A1  - Bischler, Thorsten
A1  - Förstner, Konrad U.
A1  - Sharma, Cynthia M.
A1  - Diekert, Gabriele
A1  - Schubert, Torsten
T1  - Tetrachloroethene respiration in Sulfurospirillum species is regulated by a two‐component system as unraveled by comparative genomics, transcriptomics, and regulator binding studies
JF  - MicrobiologyOpen
N2  - Energy conservation via organohalide respiration (OHR) in dehalogenating Sulfurospirillum species is an inducible process. However, the gene products involved in tetrachloroethene (PCE) sensing and signal transduction have not been unambiguously identified. Here, genome sequencing of Sulfurospirillum strains defective in PCE respiration and comparative genomics, which included the PCE‐respiring representatives of the genus, uncovered the genetic inactivation of a two‐component system (TCS) in the OHR gene region of the natural mutants. The assumption that the TCS gene products serve as a PCE sensor that initiates gene transcription was supported by the constitutive low‐level expression of the TCS operon in fumarate‐adapted cells of Sulfurospirillum multivorans. Via RNA sequencing, eight transcriptional units were identified in the OHR gene region, which includes the TCS operon, the PCE reductive dehalogenase operon, the gene cluster for norcobamide biosynthesis, and putative accessory genes with unknown functions. The OmpR‐family response regulator (RR) encoded in the TCS operon was functionally characterized by promoter‐binding assays. The RR bound a cis‐regulatory element that contained a consensus sequence of a direct repeat (CTATW) separated by 17 bp. Its location either overlapping the −35 box or 50 bp further upstream indicated different regulatory mechanisms. Sequence variations in the regulator binding sites identified in the OHR gene region were in accordance with differences in the transcript levels of the respective gene clusters forming the PCE regulon. The results indicate the presence of a fine‐tuned regulatory network controlling PCE metabolism in dehalogenating Sulfurospirillum species, a group of metabolically versatile organohalide‐respiring bacteria.
KW  - genomics
KW  - organohalide respiration
KW  - RNA sequencing
KW  - tetrachloroethene
KW  - transcriptomics
KW  - two‐component system
Y1  - 2020
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-225754
VL  - 9
IS  - 12
ER  - 
TY  - JOUR
A1  - Westermann, Alexander J.
A1  - Barquist, Lars
A1  - Vogel, Jörg
T1  - Resolving host-pathogen interactions by dual RNA-seq
JF  - PLoS Pathogens
N2  - The transcriptome is a powerful proxy for the physiological state of a cell, healthy or diseased. As a result, transcriptome analysis has become a key tool in understanding the molecular changes that accompany bacterial infections of eukaryotic cells. Until recently, such transcriptomic studies have been technically limited to analyzing mRNA expression changes in either the bacterial pathogen or the infected eukaryotic host cell. However, the increasing sensitivity of high-throughput RNA sequencing now enables “dual RNA-seq” studies, simultaneously capturing all classes of coding and noncoding transcripts in both the pathogen and the host. In the five years since the concept of dual RNA-seq was introduced, the technique has been applied to a range of infection models. This has not only led to a better understanding of the physiological changes in pathogen and host during the course of an infection but has also revealed hidden molecular phenotypes of virulence-associated small noncoding RNAs that were not visible in standard infection assays. Here, we use the knowledge gained from these recent studies to suggest experimental and computational guidelines for the design of future dual RNA-seq studies. We conclude this review by discussing prospective applications of the technique.
KW  - Medicine
KW  - RNA sequencing
KW  - Salmonellosis
KW  - Transcriptome analysis
KW  - Gene expression
KW  - Bacterial pathogens
KW  - Salmonella
KW  - Host cells
KW  - Lysis (medicine)
Y1  - 2017
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-171921
VL  - 13
IS  - 2
ER  - 
TY  - JOUR
A1  - Caliskan, Aylin
A1  - Crouch, Samantha A. W.
A1  - Giddins, Sara
A1  - Dandekar, Thomas
A1  - Dangwal, Seema
T1  - Progeria and aging — Omics based comparative analysis
JF  - Biomedicines
N2  - Since ancient times aging has also been regarded as a disease, and humankind has always strived to extend the natural lifespan. Analyzing the genes involved in aging and disease allows for finding important indicators and biological markers for pathologies and possible therapeutic targets. An example of the use of omics technologies is the research regarding aging and the rare and fatal premature aging syndrome progeria (Hutchinson-Gilford progeria syndrome, HGPS). In our study, we focused on the in silico analysis of differentially expressed genes (DEGs) in progeria and aging, using a publicly available RNA-Seq dataset (GEO dataset GSE113957) and a variety of bioinformatics tools. Despite the GSE113957 RNA-Seq dataset being well-known and frequently analyzed, the RNA-Seq data shared by Fleischer et al. is far from exhausted and reusing and repurposing the data still reveals new insights. By analyzing the literature citing the use of the dataset and subsequently conducting a comparative analysis comparing the RNA-Seq data analyses of different subsets of the dataset (healthy children, nonagenarians and progeria patients), we identified several genes involved in both natural aging and progeria (KRT8, KRT18, ACKR4, CCL2, UCP2, ADAMTS15, ACTN4P1, WNT16, IGFBP2). Further analyzing these genes and the pathways involved indicated their possible roles in aging, suggesting the need for further in vitro and in vivo research. In this paper, we (1) compare “normal aging” (nonagenarians vs. healthy children) and progeria (HGPS patients vs. healthy children), (2) enlist genes possibly involved in both the natural aging process and progeria, including the first mention of IGFBP2 in progeria, (3) predict miRNAs and interactomes for WNT16 (hsa-mir-181a-5p), UCP2 (hsa-mir-26a-5p and hsa-mir-124-3p), and IGFBP2 (hsa-mir-124-3p, hsa-mir-126-3p, and hsa-mir-27b-3p), (4) demonstrate the compatibility of well-established R packages for RNA-Seq analysis for researchers interested but not yet familiar with this kind of analysis, and (5) present comparative proteomics analyses to show an association between our RNA-Seq data analyses and corresponding changes in protein expression.
KW  - progeria
KW  - aging
KW  - omics
KW  - RNA sequencing
KW  - bioinformatics
KW  - sun exposure
KW  - HGPS
KW  - IGFBP2
KW  - ACKR4
KW  - WNT
Y1  - 2022
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-289868
SN  - 2227-9059
VL  - 10
IS  - 10
ER  - 
TY  - JOUR
A1  - Lioliou, Efthimia
A1  - Sharma, Cynthia M.
A1  - Caldelari, Isabelle
A1  - Helfer, Anne-Catherine
A1  - Fechter, Pierre
A1  - Vandenesch, François
A1  - Vogel, Jörg
A1  - Romby, Pascale
T1  - Global Regulatory Functions of the Staphylococcus aureus Endoribonuclease III in Gene Expression
JF  - PLoS Genetics
N2  - RNA turnover plays an important role in both virulence and adaptation to stress in the Gram-positive human pathogen Staphylococcus aureus. However, the molecular players and mechanisms involved in these processes are poorly understood. Here, we explored the functions of S. aureus endoribonuclease III (RNase III), a member of the ubiquitous family of double-strand-specific endoribonucleases. To define genomic transcripts that are bound and processed by RNase III, we performed deep sequencing on cDNA libraries generated from RNAs that were co-immunoprecipitated with wild-type RNase III or two different cleavage-defective mutant variants in vivo. Several newly identified RNase III targets were validated by independent experimental methods. We identified various classes of structured RNAs as RNase III substrates and demonstrated that this enzyme is involved in the maturation of rRNAs and tRNAs, regulates the turnover of mRNAs and non-coding RNAs, and autoregulates its synthesis by cleaving within the coding region of its own mRNA. Moreover, we identified a positive effect of RNase III on protein synthesis based on novel mechanisms. RNase III–mediated cleavage in the 5′ untranslated region (5′UTR) enhanced the stability and translation of cspA mRNA, which encodes the major cold-shock protein. Furthermore, RNase III cleaved overlapping 5′UTRs of divergently transcribed genes to generate leaderless mRNAs, which constitutes a novel way to co-regulate neighboring genes. In agreement with recent findings, low abundance antisense RNAs covering 44% of the annotated genes were captured by co-immunoprecipitation with RNase III mutant proteins. Thus, in addition to gene regulation, RNase III is associated with RNA quality control of pervasive transcription. Overall, this study illustrates the complexity of post-transcriptional regulation mediated by RNase III.
KW  - staphylococcus aureus
KW  - ribonucleases
KW  - messenger RNA
KW  - RNA sequencing
KW  - antisense RNA
KW  - RNA structure
KW  - RNA synthesis
KW  - RNA denaturation
Y1  - 2012
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-127219
VL  - 8
IS  - 6
ER  - 
TY  - THES
A1  - Bischler, Thorsten David
T1  - Data mining and software development for RNA-seq-based approaches in bacteria
T1  - Data-Mining und Softwareentwicklung für RNA-seq-basierte Methoden bei Bakterien
N2  - RNA sequencing (RNA-seq) has in recent years become the preferred method for gene expression analysis and whole transcriptome annotation. While initial RNA-seq experiments focused on eukaryotic messenger RNAs (mRNAs), which can be purified from the cellular ribonucleic acid (RNA) pool with relative ease, more advanced protocols had to be developed for sequencing of microbial transcriptomes. The resulting RNA-seq data revealed an unexpected complexity of bacterial transcriptomes and the requirement for specific analysis methods, which in many cases is not covered by tools developed for processing of eukaryotic data.
The aim of this thesis was the development and application of specific data analysis methods for different RNA-seq-based approaches used to gain insights into transcription and gene regulatory processes in prokaryotes.
The differential RNA sequencing (dRNA-seq) approach allows for transcriptional start site (TSS) annotation by differentiating between primary transcripts with a 5’-triphosphate (5’-PPP) and processed transcripts with a 5’-monophosphate (5’-P). This method was applied in combination with an automated TSS annotation tool to generate global trancriptome maps for Escherichia coli (E. coli) and Helicobacter pylori (H. pylori).
In the E. coli study we conducted different downstream analyses to gain a deeper understanding of the nature and properties of transcripts in our TSS map. Here, we focused especially on putative antisense RNAs (asRNAs), an RNA class transcribed from the opposite strand of known protein-coding genes with the potential to regulate corresponding sense transcripts. Besides providing a set of putative asRNAs and experimental validation of candidates via Northern analysis, we analyzed and discussed different sources of variation in RNA-seq data.
The aim of the H. pylori study was to provide a detailed description of the dRNA-seq approach and its application to a bacterial model organism. It includes information on experimental protocols and requirements for data analysis to generate a genome-wide TSS map. We show how the included TSS can be used to identify and analyze transcriptome and regulatory features and discuss challenges in terms oflibrary preparation protocols, sequencing platforms, and data analysis including manual and automated TSS annotation.
The TSS maps and associated transcriptome data from both H. pylori and E. coli were made available for visualization in an easily accessible online browser.
Furthermore, a modified version of dRNA-seq was used to identify transcriptome targets of the RNA pyrophosphohydrolase (RppH) in H. pylori. RppH initiates 5’-end-dependent degradation of transcripts by converting the 5’-PPP of primary transcripts to a 5’-P. I developed an analysis method, which uses data from complementary DNA (cDNA) libraries specific for transcripts carrying a 5’-PPP, 5’-P or both, to specifically identify transcripts modified by RppH. For this, the method assessed the 5’-phosphorylation state and cellular concentration of transcripts in rppH deletion in comparison to strains with the intact gene. Several of the identified potential RppH targets were further validated via half-life measurements and quantification of their 5’-phosphorylation state in wild-type and mutant cells. Our findings suggest an important role for RppH in post-transcriptional gene regulationin H. pylori and related organisms.
In addition, we applied two RNA-seq -based approaches, RNA immunoprecipitation followed by sequencing (RIP-seq) and cross-linking immunoprecipitation followed by sequencing (CLIP-seq), to identify transcripts bound by Hfq and CsrA, two RNA-binding proteins (RBPs) with an important role in post-transcriptional regulation.
For RIP-seq -based identification of CsrA binding regions in Campylobacter jejuni(C. jejuni), we used annotation-based analysis and, in addition, a self-developed peak calling method based on a sliding window approach. Both methods revealed flaA mRNA, encoding the major flagellin, as the main target and functional analysis of identified targets showed a significant enrichment of genes involved in flagella biosynthesis. Further experimental analysis revealed the role of flaA mRNA in post-transcriptional regulation. In comparison to RIP-seq, CLIP-seq allows mapping of RBP binding sites with a higher resolution. To identify these sites an approach called “block-based peak calling” was developed and resulting peaks were used to identify sequence and structural constraints required for interaction of Hfq and CsrA with Salmonella transcripts.
Overall, the different RNA-seq-based approaches described in this thesis together with their associated analyis pipelines extended our knowledge on the transcriptional repertoire and modes of post-transcriptional regulation in bacteria. The global TSS maps, including further characterized asRNA candidates, putative RppH targets, and identified RBP interactomes will likely trigger similar global studies in the same or different organisms or will be used as a resource for closer examination of these features.
N2  - RNA-Sequenzierung (RNA-seq) entwickelte sich in den letzten Jahren zur bevorzugten Methode für Genexpressionsanalysen und die Annotation ganzer Transkriptome. Nachdem sich erste RNA-seq-Experimente hauptsächlich mit eukaryotischen Boten-RNAs (mRNAs) beschäftigt hatten, da diese sich relativ einfach aus dem zellulären RNA-Gemisch aufreinigen lassen, war die Entwicklung von fortschrittlicheren Methoden nötig, um mikrobielle Transkriptome zu sequenzieren. Die sich daraus ergebenden RNA-seq-Daten enthüllten eine unerwartete Komplexität bakterieller Transkriptome und die Notwendigkeit der Anwendung spezifischer Analyseverfahren, welche von Tools zur Prozessierung eukaryotischer Daten häufig nicht zur Verfügung gestellt werden.
Das Ziel dieser Doktorarbeit war die Entwicklung und Anwendung spezifischer Verfahren zur Datenanalyse für verschiedene RNA-seq-basierte Methoden, um Erkenntnisse bezüglich Transkription und genregulatorischer Vorgänge bei Prokaryoten zu erlangen.
Die Differentielle-RNA-Sequenzierungsmethode (dRNA-seq) ermöglicht die Annotation von Transkriptionsstartpunkten (TSS), indem sie Primärtranskripte mit einem 5'-Triphosphat (5'-PPP) von prozessierten Transkripten mit einem 5'-Monophosphat (5'-P) unterscheidet. Diese Methode wurde in Kombination mit einem automatisierten TSS-Annotationstool zur Erstellung globaler Transkriptomkarten für Escherichia coli (E. coli) and Helicobacter pylori (H. pylori) verwendet.
In der E. coli-Studie haben wir verschiedene Folgeanalysen durchgeführt, um ein tieferes Verständnis für die Natur und Eigenschaften der in unserer Transkriptomkarte enthaltenen Transkripte zu erlangen. Das Hauptaugenmerk lag dabei auf mutmaßlichen Antisense-RNAs (asRNAs). Diese stellen eine RNA-Klasse dar, welche vom entgegengesetzten Strang von bekannten proteinkodierenden Genen transkribiert wird, und die das Potenzial hat, entsprechende Sense-Transkripte zu regulieren. Wir stellen nicht nur eine Liste mutmaßlicher asRNAs zur Verfügung, von der einige Kandidaten durch Northern Blots validiert wurden, sondern diskutierten auch von uns untersuchte Gründe für auftretende Variation bei RNA-seq-Daten.
Das Ziel der H. pylori-Studie war es, eine detaillierte Beschreibung der dRNA-seq-Methode und deren Anwendung auf einen bakteriellen Modellorganismus zur Verfügung zu stellen. Sie enthält Informationen bezüglich experimenteller Protokolle und für die Datenanalyse notwendige Schritte, zur Erstellung einer genomweiten TSS-Karte. Wir zeigen, wie die enthaltenen TSS verwendet werden können, um verschiedene Transkriptomelemente, einschließlich solcher mit regulatorischen Eigenschaften, zu identifizieren und zu analysieren. Zusätzlich diskutieren wir Probleme, welche bei der Erstellung von Sequenzierlibraries, der Verwendung von Sequenzierplattformen und bei der Datenanalyse, einschließlich manueller und automatisierter TSS-Annotation, auftreten können.
Die TSS-Karten für H. pylori und E. coli, einschließlich der damit verbundenen Transkriptomdaten, haben wir in Form eines leicht zugänglichen Online-Browsers verfügbar gemacht.
Desweiteren wurde eine modifizierte Version der dRNA-seq-Methode verwendet, um Transkripte zu identifizieren, welche von der RNA Pyrophosphohydrolase (RppH) in H. pylori gespalten werden. RppH initiiert den vom 5'-Ende abhängigen RNA-Abbau, indem sie das 5'-PPP von Primärtranskripten in ein 5'-P umwandelt. Ich habe eine Analysemethode entwickelt, welche Daten basierend auf unterschiedlichen Komplementär-DNA (cDNA)-Libraries verwendet, welche entweder spezifisch für Transkripte mit einem 5'-PPP oder einem 5'-P sind, oder beides enthalten, um spezifisch Transkripte zu indentifizieren, die durch RppH modifiziert werden. Um dies zu erreichen wurden der 5'-Phosphorylierungsstatus und die zelluläre Konzentration der Transkripte zwischen einer rppH-Deletionsmutante und Stämmen mit intaktem Gen verglichen. Weiterhin wurden mehrere der identifizierten, von RppH gespaltenen Transkripte durch Messung ihrer Halbwertszeit und Quantifizierung ihres 5'-Phosphorylierungsstatus bei Wildtyp- und mutierten Zellen validiert. Unsere Ergebnisse lassen auf eine wichtige Rolle von RppH bei der Genregulation in H. pylori und verwandten Organismen schließen.
Zusätzlich haben wir zwei weitere RNA-seq-basierte Methoden namens RNA-Immunpräzipitation gefolgt von RNA-Sequenzierung (RIP-seq) und Quervernetzung und Immunpräzipitation gefolgt von RNA-Sequenzierung (CLIP-seq) verwendet, um Transkripte zu identifizieren, welche von Hfq und CsrA gebunden werden, zwei RNA-Bindeproteinen (RBPs), die eine wichtige Rolle bei posttranskriptionaler Regulation spielen.
Zur RIP-seq-basierten Identifikation von CsrA-Binderegionen bei Campylobacter jejuni (C. jejuni) haben wir eine annotationsbasierte Analyse und zusätzlich eine eigens entwickelte Peak-Bestimmungsmethode verwendet. Beide Methoden haben die flaA mRNA, welche das Hauptflagellin kodiert, als stärksten Bindepartner identifiziert. Die Funktionale-Anreicherungsanalyse hat außerdem eine Anreicherung von Genen ergeben, welche für die Flagellenbiosynthese von Bedeutung sind.
Im Vergleich zu RIP-seq ermöglicht CLIP-seq eine höhere Auflösung bei der Kartografierung von Bindestellen. Um diese Stellen zu identifizieren wurde eine Methode mit der Bezeichnung ``block-based peak calling'' entwickelt, und die daraus resultierenden Peaks wurden verwendet, um sequenz- und strukturabhängige Bedingungen zu bestimmen, die bei Salmonella für die Interaktion von Transkripten mit Hfq und CsrA notwendig sind.
Insgesamt betrachtet haben die verschiedenen RNA-seq-basierten Methoden, welche in dieser Doktorarbeit beschrieben wurden, in Kombination mit den damit verbundenen Analysepipelines, unser Verständnis des transkriptionellen Repertoires und der Art und Weise, wie posttranskriptionelle Regulation bei Bakterien abläuft, erweitert. Die globalen TSS-Karten, einschließlich der charakterisierten asRNA-Kandidaten, die mutmaßlich von RppH gespaltenen Transkripte und die identifizierten RBP-Interaktome werden höchstwahrscheinlich zur Durchführung ähnlicher Studien bei den gleichen oder anderen Organismen führen, oder können als Grundlage für eine detailliertere Untersuchung dieser Elemente verwendet werden.
KW  - Bakterien
KW  - RNA sequencing
KW  - Bioinformatics
KW  - Bacteria
KW  - Transcriptome
KW  - Post-transcriptional regulation
KW  - RNA-binding proteins
KW  - Sequenzanalyse <Chemie>
KW  - RNS
Y1  - 2018
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-166108
ER  -