Wang Zuo, Wang Shuang, Bi Yi, Boiti Alessandra, Zhang Shengxiang, Vallone Daniela, Lan Xianyong, Foulkes Nicholas S, Zhao Haiyu
School of Life Sciences, Gansu Key Laboratory of Biomonitoring and Bioremediation for Environmental Pollution, Lanzhou University, Lanzhou, China.
Institute of Biological and Chemical Systems, Biological Information Processing (IBCS-BIP), Karlsruhe Institute of Technology (KIT), Eggenstein-Leopoldshafen, Germany.
PLoS Genet. 2025 Jan 8;21(1):e1011545. doi: 10.1371/journal.pgen.1011545. eCollection 2025 Jan.
A key property of the circadian clock is that it is reset by light to remain synchronized with the day-night cycle. An attractive model to explore light input to the circadian clock in vertebrates is the zebrafish. Circadian clocks in zebrafish peripheral tissues and even zebrafish-derived cell lines are entrainable by direct light exposure thus providing unique insight into the function and evolution of light regulatory pathways. Our previous work has revealed that light-induced gene transcription is a key step in the entrainment of the circadian clock as well as enabling the more general adaptation of zebrafish cells to sunlight exposure. However, considerable evidence points to post-transcriptional regulatory mechanisms, notably microRNAs (miRNAs), playing an essential role in shaping dynamic changes in mRNA levels. Therefore, does light directly impact the function of miRNAs? Are there light-regulated miRNAs and if so, which classes of mRNA do they target? To address these questions, we performed a complete sequencing analysis of light-induced changes in the zebrafish transcriptome, encompassing small non-coding RNAs as well as mRNAs. Importantly, we identified sets of light-regulated miRNAs, with many regulatory targets representing light-inducible mRNAs including circadian clock genes and genes involved in redox homeostasis. We subsequently focused on the light-responsive miR-204-3-3p and miR-430a-3p which are predicted to regulate the expression of cryptochrome genes (cry1a and cry1b). Luciferase reporter assays validated the target binding of miR-204-3-3p and miR-430a-3p to the 3'UTRs of cry1a and cry1b, respectively. Furthermore, treatment with mimics and inhibitors of these two miRNAs significantly affected the dynamic expression of their target genes but also other core clock components (clock1a, bmal1b, per1b, per2, per3), as well as the rhythmic locomotor activity of zebrafish larvae. Thus, our identification of light-responsive miRNAs reveals new intricacy in the multi-level regulation of the circadian clockwork by light.
昼夜节律钟的一个关键特性是它会被光重置，从而与昼夜循环保持同步。探索脊椎动物昼夜节律钟的光输入的一个有吸引力的模型是斑马鱼。斑马鱼外周组织甚至斑马鱼衍生的细胞系中的昼夜节律钟可通过直接光照进行同步，从而为光调节途径的功能和进化提供独特的见解。我们之前的研究表明，光诱导的基因转录是昼夜节律钟同步的关键步骤，也是使斑马鱼细胞更普遍地适应阳光照射的关键。然而，大量证据表明转录后调控机制，尤其是微小RNA（miRNA），在塑造mRNA水平的动态变化中起着至关重要的作用。那么，光是否直接影响miRNA的功能？是否存在受光调节的miRNA，如果存在，它们靶向哪类mRNA？为了解决这些问题，我们对斑马鱼转录组中光诱导的变化进行了全面的测序分析，包括小非编码RNA和mRNA。重要的是，我们鉴定出了受光调节的miRNA集合，许多调控靶点代表光诱导的mRNA，包括昼夜节律钟基因和参与氧化还原稳态的基因。随后，我们聚焦于光响应性miR-204-3-3p和miR-430a-3p，它们被预测可调节隐花色素基因（cry1a和cry1b）的表达。荧光素酶报告基因检测分别验证了miR-204-3-3p和miR-430a-3p与cry1a和cry1b的3'UTR的靶标结合。此外，用这两种miRNA的模拟物和抑制剂处理不仅显著影响其靶基因的动态表达，还影响其他核心生物钟成分（clock1a、bmal1b、per1b、per2、per3）以及斑马鱼幼虫的节律性运动活动。因此，我们对光响应性miRNA的鉴定揭示了光对昼夜节律钟多层次调控中的新复杂性。
