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Transcriptional and posttranscriptional regulation of the microRNA pathway by 5-lipoxygenase

Hedrich, Stella Andrea (2021):
Transcriptional and posttranscriptional regulation of the microRNA pathway by 5-lipoxygenase. (Publisher's Version)
Darmstadt, Technische Universität,
DOI: 10.26083/tuprints-00009097,
[Ph.D. Thesis]

Abstract

MicroRNAs (miRNA) are prominent players of the gene silencing pathway that control the majority of human protein-coding genes and thus are involved in almost every physiological process. The biosynthesis of these important posttranscriptional regulators of gene expression needs to be tightly controlled. In addition to the transcriptional regulation of miRNAs, it becomes more and more evident that the miRNA processing can also be modulated. For instance, the enzyme 5-lipoxygenase (5-LO), which is mainly known for its catalyzing properties in the biosynthesis of inflammatory leukotrienes and specialized pro-resolving mediators, and whose expression is also controlled by miRNAs, interferes with the miRNA pathway through its interaction with Dicer, a key enzyme of the miRNAs’ processing. Thus, this thesis aims to further investigate the interaction of 5-LO and Dicer, as well as its functional impact on the miRNA processing. The 5-LO binding to Dicer was previously characterized using in vitro methods. Performing proximity ligation assays, that allow monitoring of endogenous protein complexes in their natural environment, demonstrated an in situ interaction of the target proteins in Mono Mac 6 cells, a human monocytic cell line that exerts a heavily increased 5-LO protein expression upon stimulation with TGF-β, calcitriol, and LPS or zymosan. Sequencing of small noncoding RNAs identified the clustered miRNAs miR-99b, let-7e, and miR-125a as potential 5-LO targets. Real-time quantitative PCR experiments were used to show that 5-LO acts both as a transcriptional as well as a posttranscriptional regulator by modifying both processing levels in opposing directions. More precisely, 5-LO exerts stimulatory effects on the transcription of the primary miRNA encoding for the miR-99b/let-7e/miR-125a cluster, but in turn also exerts inhibitory effects on the Dicer-mediated cleavage of the let-7e precursor. The processing of the other clustered miRNAs, namely miR-99b and miR-125a, is unaffected or just slightly enhanced, revealing a pre-miRNA specific and dose-dependent 5-LO effect, which was confirmed by performing in vitro Dicer assays. The mutually overlapping effects result in an upregulation of mature miR-99b and miR-125a with simultaneously unchanged let-7e levels. Since the cluster members exert different biological functions, especially in the context of stem cells, these observations might describe a mechanism to modulate the expression of each member within miRNA clusters individually. It is worth noting that LPS and zymosan share the exceptional characteristic of contrastingly modulating one pathway at different regulatory levels. Real-time quantitative PCR experiments were utilized to demonstrate that whenever the TLR activators LPS or zymosan stimulate the transcription of a miRNA they also inhibit its subsequent processing, resulting in increased levels of the NF-κB-dependent TLR-response miRNAs miR-146a, miR-21, miR-125a, and let-7e, all of which function as negative feedback regulators of the TLR signaling. This precise regulation of the miRNA processing by LPS and zymosan, which seems to be independent of 5-LO, provides further evidence for the tight interconnections between miRNAs and the innate immune responses mediated via TLR signaling. In conclusion, the opposing modulation at multiple levels by a single determinant within one pathway could potentially be a generally valid principle which allows fine tuning of the miRNA expression through opposing effects that dampen each other. Furthermore, the findings gained within the course of this study contribute to characterizing 5-LO’s role within the miRNA pathway.

Item Type: Ph.D. Thesis
Erschienen: 2021
Creators: Hedrich, Stella Andrea
Status: Publisher's Version
Title: Transcriptional and posttranscriptional regulation of the microRNA pathway by 5-lipoxygenase
Language: English
Abstract:

MicroRNAs (miRNA) are prominent players of the gene silencing pathway that control the majority of human protein-coding genes and thus are involved in almost every physiological process. The biosynthesis of these important posttranscriptional regulators of gene expression needs to be tightly controlled. In addition to the transcriptional regulation of miRNAs, it becomes more and more evident that the miRNA processing can also be modulated. For instance, the enzyme 5-lipoxygenase (5-LO), which is mainly known for its catalyzing properties in the biosynthesis of inflammatory leukotrienes and specialized pro-resolving mediators, and whose expression is also controlled by miRNAs, interferes with the miRNA pathway through its interaction with Dicer, a key enzyme of the miRNAs’ processing. Thus, this thesis aims to further investigate the interaction of 5-LO and Dicer, as well as its functional impact on the miRNA processing. The 5-LO binding to Dicer was previously characterized using in vitro methods. Performing proximity ligation assays, that allow monitoring of endogenous protein complexes in their natural environment, demonstrated an in situ interaction of the target proteins in Mono Mac 6 cells, a human monocytic cell line that exerts a heavily increased 5-LO protein expression upon stimulation with TGF-β, calcitriol, and LPS or zymosan. Sequencing of small noncoding RNAs identified the clustered miRNAs miR-99b, let-7e, and miR-125a as potential 5-LO targets. Real-time quantitative PCR experiments were used to show that 5-LO acts both as a transcriptional as well as a posttranscriptional regulator by modifying both processing levels in opposing directions. More precisely, 5-LO exerts stimulatory effects on the transcription of the primary miRNA encoding for the miR-99b/let-7e/miR-125a cluster, but in turn also exerts inhibitory effects on the Dicer-mediated cleavage of the let-7e precursor. The processing of the other clustered miRNAs, namely miR-99b and miR-125a, is unaffected or just slightly enhanced, revealing a pre-miRNA specific and dose-dependent 5-LO effect, which was confirmed by performing in vitro Dicer assays. The mutually overlapping effects result in an upregulation of mature miR-99b and miR-125a with simultaneously unchanged let-7e levels. Since the cluster members exert different biological functions, especially in the context of stem cells, these observations might describe a mechanism to modulate the expression of each member within miRNA clusters individually. It is worth noting that LPS and zymosan share the exceptional characteristic of contrastingly modulating one pathway at different regulatory levels. Real-time quantitative PCR experiments were utilized to demonstrate that whenever the TLR activators LPS or zymosan stimulate the transcription of a miRNA they also inhibit its subsequent processing, resulting in increased levels of the NF-κB-dependent TLR-response miRNAs miR-146a, miR-21, miR-125a, and let-7e, all of which function as negative feedback regulators of the TLR signaling. This precise regulation of the miRNA processing by LPS and zymosan, which seems to be independent of 5-LO, provides further evidence for the tight interconnections between miRNAs and the innate immune responses mediated via TLR signaling. In conclusion, the opposing modulation at multiple levels by a single determinant within one pathway could potentially be a generally valid principle which allows fine tuning of the miRNA expression through opposing effects that dampen each other. Furthermore, the findings gained within the course of this study contribute to characterizing 5-LO’s role within the miRNA pathway.

Place of Publication: Darmstadt
Collation: X, 94 Seiten
Divisions: 10 Department of Biology
10 Department of Biology > Synthetic Genetic Circuits
Date Deposited: 08 Feb 2021 10:46
DOI: 10.26083/tuprints-00009097
Official URL: https://tuprints.ulb.tu-darmstadt.de/9097
URN: urn:nbn:de:tuda-tuprints-90977
Referees: Süß, Prof. Dr. Beatrix ; Steinhilber, Prof. Dr. Dieter
Refereed / Verteidigung / mdl. Prüfung: 12 August 2019
Alternative Abstract:
Alternative abstract Language

MicroRNAs (miRNA) sind bedeutsame Akteure des Gen-Silencing Signalwegs, da sie die Mehrzahl der humanen Protein-kodierenden Gene kontrollieren und demzufolge in nahezu jeden physiologischen Prozess involviert sind. Die Biosynthese dieser wichtigen posttranskriptionellen Regulatoren der Genexpression bedarf daher einer strikten Kontrolle. Wissenschaftliche Erkenntnisse zeigen, dass nicht nur die Transkription der miRNAs reguliert werden kann, sondern auch deren Prozessierung. Beispielsweise interagiert das Enzym die 5-Lipoxygenase (5-LO), welches vor allem für seine katalytische Aktivität in der Biosynthese der inflammatorischen Leukotriene und SPMs (specialized pro-resolving mediators) bekannt ist und dessen Expression ebenfalls durch miRNAs reguliert wird, mit Dicer. Da es sich bei Dicer um ein Schlüsselenzym der miRNA-Prozessierung handelt, beeinflusst die 5-LO möglicherweise durch die Interaktion mit Dicer die Synthese der miRNAs. Die vorliegende Doktorarbeit dient dem Zweck, die Interaktion zwischen 5-LO und Dicer, sowie deren funktionalen Einfluss auf die miRNA-Biosynthese näher zu untersuchen Die Bindung von 5-LO zu Dicer wurde bereits unter Verwendung von in vitro Methoden charakterisiert. Die Durchführung von Proximity Ligation Assays, eine Methode, welche das Monitoring endogener Proteinkomplexe in ihrer natürlichen Umgebung ermöglicht, bestätigte eine in situ Interaktion zwischen den Zielproteinen in Mono Mac 6 (MM6) Zellen. Bei den MM6-Zellen handelt es sich um eine humane monozytische Zelllinie, welche einen starken Anstieg der 5-LO-Proteinexpression nach Stimulierung mit TGF-β, Calcitriol und LPS oder Zymosan zeigt. Die Sequenzierung kurzer nicht-kodierender RNAs identifizierte das miRNA-Cluster miR-99b, let-7e und miR-125a als ein potenzielles Ziel der 5-LO. Durch die Durchführung der quantitativen Echtzeit-PCR konnte gezeigt werden, dass die 5-LO sowohl als transkriptioneller als auch als posttranskriptioneller Regulator agiert, indem das Enzym beide Prozessierungsebenen in unterschiedliche Richtungen modifiziert. Einerseits stimuliert die 5-LO das Primärtranskript, welches für das miR-99b/let-7e/miR-125a Cluster kodiert, andererseits inhibiert das Enzym die Dicer-vermittelte Prozessierung des let-7e Vorläufers. Die Reifung der anderen miRNAs, miR-99b und miR-125a, wird nicht beeinflusst beziehungsweise leicht stimuliert. Dies verdeutlicht, dass die 5-LO pre-miRNA-spezifische sowie dosisabhängige Effekte ausübt, welche durch die Durchführung von in vitro Dicer Assays bestätigt werden konnten. Die gegenläufigen und sich überschneidenden Effekte resultieren in einer Hochregulierung der reifen miRNAs miR-99b und miR-125a mit gleichzeitig unveränderten let-7e-Spiegeln. Angesichts der divergierenden biologischen Funktionen der beschriebenen miRNAs, insbesondere im Kontext von Stammzellen, beschreiben diese Beobachtungen einen Mechanismus zur individuellen Regulierung der Expression einzelner miRNAs innerhalb eines Clusters. Es ist bemerkenswert, dass LPS und Zymosan ebenso wie 5-LO die außergewöhnliche Fähigkeit besitzen, einen Signalweg auf verschiedenen Ebenen gegenläufig zu regulieren. Die Ergebnisse quantitativer Echtzeit-PCR-Experimente zeigen, dass, wann immer die TLR-Aktivatoren LPS oder Zymosan die Transkription einer miRNA stimulieren, sie nachfolgend deren Prozessierung hemmen. Dies resultiert in einer erhöhten Expression der NF-κB-abhängigen und TLR-responsiven miRNAs miR-146a, miR-21, miR-125a und let-7e, welche als negative Feedback-Regulatoren des TLR-Signalwegs fungieren. Diese präzise und scheinbar von 5-LO-unabhängige Regulation der miRNA-Prozessierung durch LPS und Zymosan verdeutlicht die enge Verflechtung zwischen miRNAs und der über den TLR-Signalweg vermittelten angeborenen Immunantwort. Zusammenfassend kann festgehalten werden, dass die gegensätzliche Modulation auf multiplen Ebenen durch einen einzelnen Akteur innerhalb eines Signalwegs ein allgemein gültiges Prinzip darstellen könnte, welches die Feinregulierung der miRNA-Expression durch gegenläufige Effekte, die sich gegenseitig abmildern, erlaubt. Die anhand dieser Doktorarbeit gewonnenen Erkenntnisse tragen zur Charakterisierung der Rolle der 5-LO innerhalb des miRNA-Signalwegs bei.

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