TY - THES A1 - Dugar, Gaurav T1 - Comparative transcriptomics and post-transcriptional regulation in \(Campylobacter\) \(jejuni\) T1 - Vergleichende Transkriptomanalysen und posttranskriptionelle Regulierung in \(Campylobacter\) \(jejuni\) N2 - The transcriptome is defined as the set of all RNA molecules transcribed in a cell. These include protein-coding messenger RNAs (mRNAs) as well as non-coding RNAs, such as ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), and small non-coding RNAs (sRNAs). sRNAs are known to play an important role in regulating gene expression and virulence in pathogens. In this thesis, the transcriptome of the food-borne pathogen Campylobacter jejuni was characterized at single nucleotide resolution by use of next-generation sequencing approaches. The first genome of a C. jejuni strain was published in the year 2000. However, its transcriptome remained uncharacterized at large. C. jejuni can survive in a variety of ecological niches and hosts. However, how strain-specific transcriptional changes contribute to such adaptation is not known. In this study, the global transcriptome maps of four closely related C. jejuni strains were defined using a differential RNA-seq (dRNA-seq) approach. This analysis also included a novel automated method to annotate the transcriptional start sites (TSS) at a genome-wide scale. Next, the transcriptomes of four strains were simultaneously mapped and compared by the use of a common coordinate system derived from whole-genome alignment, termed as SuperGenome. This approach helped to refine the promoter maps by comparison of TSS within strains. Most of the TSS were found to be conserved among all four strains, but some single-nucleotide-polymorphisms (SNPs) around promoter regions led to strain-specific transcriptional output. Most of these SNPs altered transcription only slightly, but some others led to a complete abrogation of transcription leading to differential molecular phenotypes. These in turn might help the strains to adapt to their specific host or microniche. The transcriptome also unveiled a plethora of sRNAs, some of which were conserved among the four strains while others were strain specific. Furthermore, a Cas9-dependent minimal type-II CRISPR-Cas system with only three Cas genes and multiple promoters to drive the transcription of the CRISPR locus was also characterized in C. jejuni using the dRNA-seq dataset. Apart from sRNAs, the role of global RNA binding proteins (RBPs) is also unclear in C. jejuni. Aided by the global transcriptome data, the role of RBPs in post-transcriptional regulation of C. jejuni was studied at a global scale. Two of the most widely studied RNA binding proteins in bacteria are Hfq and CsrA. The RNA interactome of the translational regulator CsrA was defined using another global deep-sequencing technique that combines co-immunoprecipitation (coIP) with RNA sequencing (RIP-seq). Using this interactome dataset, the direct targets of this widespread global post-transcriptional regulator were defined, revealing a significant enrichment for mRNAs encoding genes involved in flagella biosynthesis. Unlike Gammaproteobacteria, where sRNAs such as CsrB/C, antagonize CsrA activity, no sRNAs were enriched in the CsrA-coIP in C. jejuni, indicating absence of any sRNA antagonists and novel modes of CsrA activity regulation. Instead, the CsrA regulatory pathway revealed flaA mRNA, encoding the major flagellin, as a dual-function mRNA. flaA mRNA was the main target of CsrA but it also served to antagonize CsrA activity along with the protein antagonist FliW previously identified in the Gram-positive bacterium Bacillus subtilis. Furthermore, this regulatory mRNA was also shown in this thesis to localize to the poles of elongating C. jejuni cells in a translation-dependent manner. It was also shown that this localization is dependent on the CsrA-FliW regulon, which controls the translation of flaA mRNA. The role and mechanism of flaA mRNA localization or mRNA localization in general is not yet clear in bacteria when compared to their eukaryotic counterparts. Overall, this study provides first insights into riboregulation of the bacterial pathogen C. jejuni. The work presented in this thesis unveils several novel modes of riboregulation in C. jejuni, which could be applicable more generally. Moreover, this study also lays out several unsolved intriguing questions, which may pave the way for interesting studies to come. N2 - Das Transkriptom ist definiert als die Summe aller RNA-Moleküle, die in einer Zelle transkribiert werden. Hierzu gehören sowohl protein-kodierende Boten-RNAs (mRNAs für „messenger RNAs“), als auch nicht-kodierende RNAs, wie ribosomale RNAs (rRNAs), transfer RNAs (tRNAs) und kleine nicht-kodierende RNAs (sRNAs für „small RNAs“). Diese sRNAs spielen eine wichtige Rolle in der Regulierung von Genexpression und Virulenz von Pathogenen. In der vorliegenden Arbeit wurde das Transkriptom des Lebensmittelkeims Campylobacter jejuni mit Hilfe von Next-Generation-Sequencing-Methoden charakterisiert, welche eine Auflösung des Transkriptoms auf Einzelnukleotid-Ebene ermöglichen. Obwohl eine erste Genomsequenz für C. jejuni bereits im Jahr 2000 veröffentlicht wurde, war das Transkriptom bisher größtenteils uncharakterisiert. C. jejuni besitzt die Fähigkeit in vielen ökologischen Nischen und Wirten überleben zu können. Es ist jedoch bislang unbekannt, wie stammspezifische Veränderungen des Transkriptoms zu dieser Adaption beitragen. Mittels eines differenziellen RNA-Sequenzierungsansatzes wurden in dieser Arbeit globale Transkriptomkarten von vier nahverwandten C. jejuni Stämmen erstellt. Diese Analyse beinhaltet auch eine neue automatisierte Methode zur genomweiten Identifizierung von Transkriptionsstartstellen (TSS). Anschließend wurde aus den Genomsequenzen der vier Campylobacter Stämme ein SuperGenom erstellt. Dieses wiederum diente als Referenz, anhand dessen die Transkriptome kartiert und miteinander verglichen werden konnten. Dieser Ansatz ermöglichte eine verfeinerte Kartierung der Promotoren mittels des Vergleichs verschiedener Stämme. Die meisten TSS waren innerhalb der vier Stämme konserviert. Allerdings kam es durch SNPs („single-nucleotide polymorphisms“) in den Promoterregionen zu stammspezifischem Transkriptoutput. Die meisten dieser SNPs hatten nur geringe Veränderungen der Transkription zur Folge. Manche jedoch führten zu einem kompletten Verlust der Transkription und damit zu verschiedenen molekularen Phänotypen. Diese wiederum könnten es den verschiedenen Stämmen ermöglichen, sich an ihre spezifische Wirts- oder Mikronische anzupassen. Das Transkriptom wies auch eine Fülle von sRNAs auf, von denen manche in allen vier Stämmen konserviert, andere jedoch stammspezifisch waren. Zudem wurde mittels des C. jejuni-dRNA-seq-Datensatzes ein minimales Cas9-abhängiges CRISPR-Cas-System des Typs II entdeckt. Dieses beinhaltet lediglich drei Cas-Gene, jedoch mehrere Promotoren, die die Expression des CRISPR-Lokus antreiben. Neben der Funktion von sRNAs ist auch die Rolle globaler RNA-Bindeproteine (RBPs) in C. jejuni weitestgehend unklar. Mithilfe der Transkriptomdaten wurde die Rolle von RBPs in der posttranskriptionellen Regulierung in C. jejuni untersucht. Zwei der am besten untersuchten RNA-Bindeproteine in Bakterien sind Hfq und CsrA. Das RNA-Interaktom des Translationsregulators CsrA wurde mittels eines weiteren globalen Deep-Squencing-Ansatzes definiert. Bei dieser Methode werden Coimmunopräzipitation (coIP) und RNA-Sequenzierung zum so genannten RIP-seq kombiniert. Mithilfe dieses Interaktionsdatensatzes wurden die Zielgene dieses weitverbreiteten, globalen posttranskriptionellen Regulators definiert. Hierbei wurde eine signifikante Anreicherung von mRNAs, die in die Biosynthese von Flagellen involviert sind, erkennbar. Anders als in Gammaproteobakterien, in denen sRNAs wie CsrB und CsrC die CsrA-Aktivität antagonisieren, wurden in C. jejuni keine sRNAs in der CsrA-CoIP angereichert. Dies deutet auf das Fehlen jeglicher sRNA-Antagonisten, und damit auf eine neue Art der CsrA-Aktivitätskontrolle hin. Anstelle der sRNAs wurde die flaA mRNA, welche für das Hauptflagellin kodiert, als mRNA mit dualer Funktion identifiziert. Sie ist zum einen das Hauptzielgen von CsrA, fungiert aber gleichzeitig, zusammen mit dem Protein FliW, als Antagonist von CsrA. FliW wurde bereits zuvor in dem Grampositiven Bakterium Bacillus subtilis identifiziert. In dieser Arbeit konnte zudem gezeigt werden, dass die regulatorische flaA mRNA translationsabhängig an den Polen der wachsenden C. jejuni-Zellen lokalisiert ist. Außerdem war zu erkennen, dass diese Lokalisierung abhängig von dem CsrA-FliW-Regulon stattfindet, welches die Translation der flaA-mRNA kontrolliert. Im Gegensatz zu Eukaryoten ist die Rolle, die die Lokalisation der flaA-mRNA, oder bakterieller mRNA im Allgemeinen, spielt, sowie der Mechanismus, der zu dieser Lokalisierung führt, bisher noch unklar. Zusammenfassend ermöglicht diese Arbeit einen ersten Einblick in die Riboregulierung des bakteriellen Pathogens C. jejuni. Es konnten einige neue Mechanismen dieser Art der Regulierung aufgedeckt werden, welche auch allgemeine Gültigkeit finden könnten. Zudem werden in dieser Arbeit neue, faszinierende Fragen aufgeworfen, die den Weg für weitere interessante Studien bereiten. KW - Post-transcriptional regulation KW - Transcriptome KW - SNPs KW - CsrA KW - Campylobacter jejuni KW - Transkriptom KW - Posttranskriptionelle Regulation Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-146180 ER - TY - JOUR A1 - da Cruz, Irene A1 - Rodríguez-Casuriaga, Rosana A1 - Santiñaque, Frederico F. A1 - Farías, Joaquina A1 - Curti, Gianni A1 - Capoano, Carlos A. A1 - Folle, Gustavo A. A1 - Benavente, Ricardo A1 - Sotelo-Silveira, José Roberto A1 - Geisinger, Adriana T1 - Transcriptome analysis of highly purified mouse spermatogenic cell populations: gene expression signatures switch from meiotic-to postmeiotic-related processes at pachytene stage JF - BMC Genomics N2 - Background Spermatogenesis is a complex differentiation process that involves the successive and simultaneous execution of three different gene expression programs: mitotic proliferation of spermatogonia, meiosis, and spermiogenesis. Testicular cell heterogeneity has hindered its molecular analyses. Moreover, the characterization of short, poorly represented cell stages such as initial meiotic prophase ones (leptotene and zygotene) has remained elusive, despite their crucial importance for understanding the fundamentals of meiosis. Results We have developed a flow cytometry-based approach for obtaining highly pure stage-specific spermatogenic cell populations, including early meiotic prophase. Here we combined this methodology with next generation sequencing, which enabled the analysis of meiotic and postmeiotic gene expression signatures in mouse with unprecedented reliability. Interestingly, we found that a considerable number of genes involved in early as well as late meiotic processes are already on at early meiotic prophase, with a high proportion of them being expressed only for the short time lapse of lepto-zygotene stages. Besides, we observed a massive change in gene expression patterns during medium meiotic prophase (pachytene) when mostly genes related to spermiogenesis and sperm function are already turned on. This indicates that the transcriptional switch from meiosis to post-meiosis takes place very early, during meiotic prophase, thus disclosing a higher incidence of post-transcriptional regulation in spermatogenesis than previously reported. Moreover, we found that a good proportion of the differential gene expression in spermiogenesis corresponds to up-regulation of genes whose expression starts earlier, at pachytene stage; this includes transition protein-and protamine-coding genes, which have long been claimed to switch on during spermiogenesis. In addition, our results afford new insights concerning X chromosome meiotic inactivation and reactivation. Conclusions This work provides for the first time an overview of the time course for the massive onset and turning off of the meiotic and spermiogenic genetic programs. Importantly, our data represent a highly reliable information set about gene expression in pure testicular cell populations including early meiotic prophase, for further data mining towards the elucidation of the molecular bases of male reproduction in mammals. KW - Spermatogenesis KW - Transcriptome KW - RNAseq KW - Flow cytometry Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-164574 VL - 17 ER - TY - JOUR A1 - Babski, Julia A1 - Haas, Karina A. A1 - Näther-Schindler, Daniela A1 - Pfeiffer, Friedhelm A1 - Förstner, Konrad U. A1 - Hammelmann, Matthias A1 - Hilker, Rolf A1 - Becker, Anke A1 - Sharma, Cynthia M. A1 - Marchfelder, Anita A1 - Soppa, Jörg T1 - Genome-wide identification of transcriptional start sites in the haloarchaeon Haloferax volcanii based on differential RNA-Seq (dRNA-Seq) JF - BMC Genomics N2 - Background Differential RNA-Seq (dRNA-Seq) is a recently developed method of performing primary transcriptome analyses that allows for the genome-wide mapping of transcriptional start sites (TSSs) and the identification of novel transcripts. Although the transcriptomes of diverse bacterial species have been characterized by dRNA-Seq, the transcriptome analysis of archaeal species is still rather limited. Therefore, we used dRNA-Seq to characterize the primary transcriptome of the model archaeon Haloferax volcanii. Results Three independent cultures of Hfx. volcanii grown under optimal conditions to the mid-exponential growth phase were used to determine the primary transcriptome and map the 5′-ends of the transcripts. In total, 4749 potential TSSs were detected. A position weight matrix (PWM) was derived for the promoter predictions, and the results showed that 64 % of the TSSs were preceded by stringent or relaxed basal promoters. Of the identified TSSs, 1851 belonged to protein-coding genes. Thus, fewer than half (46 %) of the 4040 protein-coding genes were expressed under optimal growth conditions. Seventy-two percent of all protein-coding transcripts were leaderless, which emphasized that this pathway is the major pathway for translation initiation in haloarchaea. A total of 2898 of the TSSs belonged to potential non-coding RNAs, which accounted for an unexpectedly high fraction (61 %) of all transcripts. Most of the non-coding TSSs had not been previously described (2792) and represented novel sequences (59 % of all TSSs). A large fraction of the potential novel non-coding transcripts were cis-antisense RNAs (1244 aTSSs). A strong negative correlation between the levels of antisense transcripts and cognate sense mRNAs was found, which suggested that the negative regulation of gene expression via antisense RNAs may play an important role in haloarchaea. The other types of novel non-coding transcripts corresponded to internal transcripts overlapping with mRNAs (1153 iTSSs) and intergenic small RNA (sRNA) candidates (395 TSSs). Conclusion This study provides a comprehensive map of the primary transcriptome of Hfx. volcanii grown under optimal conditions. Fewer than half of all protein-coding genes have been transcribed under these conditions. Unexpectedly, more than half of the detected TSSs belonged to several classes of non-coding RNAs. Thus, RNA-based regulation appears to play a more important role in haloarchaea than previously anticipated. KW - Archaea KW - dRNA-Seq KW - Promoter KW - Non-coding RNAs KW - sRNA KW - Haloferax volcanii KW - Transcriptome KW - Leaderless transcript KW - Antisense RNA Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-164553 VL - 17 IS - 629 ER -