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

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.