A miR-219-5p-bmal1b negative feedback loop contributes to circadian regulation in zebrafish

Area of Science:

• * Circadian Biology
• * Molecular Neuroscience
• * Zebrafish Model Organism

Background:

• * MicroRNAs (miRNAs) are key post-transcriptional regulators of gene expression involved in various biological processes.
• * Circadian rhythms, the endogenous ~24-hour cycles, govern physiological and behavioral patterns.
• * The role of miRNAs in regulating circadian rhythms, particularly in zebrafish, remained largely unexplored.

Purpose of the Study:

• * To investigate the involvement of specific microRNAs (miRNAs) in the regulation of circadian rhythms in zebrafish.
• * To elucidate the molecular mechanisms by which miRNAs influence circadian clock function in the zebrafish brain.

Main Methods:

• * Quantitative real-time PCR (qRT-PCR) to assess miRNA expression patterns.
• * Gene deletion and knockdown techniques (e.g., CRISPR/Cas9, antisense oligonucleotides) to study functional consequences.
• * RNA immunoprecipitation (RIP) assays, miRNA pull-down assays, and luciferase reporter assays to confirm target interactions.
• * Locomotor activity monitoring and transcriptome analysis (RNA-seq) to evaluate circadian phenotypes and molecular changes.

Main Results:

• * Identified rhythmic expression of mature miR-219-5p and its precursors (mir-219-1, mir-219-2, mir-219-3) in specific zebrafish brain regions (TeO, CCe, CC).
• * Demonstrated that mir-219-1 and mir-219-2 are clock-regulated, while mir-219-3 is light-regulated.
• * Showed that disruption of mir-219 genes or knockdown of miR-219-5p shortens locomotor rhythm periods and upregulates bmal1b.
• * Confirmed that miR-219-5p directly targets and inhibits bmal1b expression by binding to its 3' untranslated region (3'UTR).
• * Transcriptome analysis suggested roles for miR-219-5p in phototransduction and neuroactive ligand-receptor interactions.

Conclusions:

• * The mir-219 gene cluster is differentially regulated by the circadian clock and light cues in the zebrafish brain.
• * miR-219-5p plays a critical role in circadian regulation by targeting and inhibiting bmal1b, a core clock component.
• * Established a novel miR-219-5p-bmal1b negative feedback loop within the zebrafish circadian circuitry.
• * Highlights the importance of miRNA-mediated regulation in maintaining robust circadian rhythms.