Summary of RNA-seq read counts.

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.

昼夜节律时钟（circadian clock）的核心特性之一，是可通过光线重置以维持与昼夜周期的同步。斑马鱼是探索脊椎动物昼夜节律时钟光输入通路的极具吸引力的模型生物。斑马鱼外周组织乃至其来源细胞系的昼夜节律时钟，均可通过直接光照实现同步化，这为解析光调控通路的功能与演化提供了独特研究视角。我们此前的研究揭示，光诱导的基因转录是昼夜节律时钟同步化的关键步骤，同时也能帮助斑马鱼细胞更广泛地适应阳光暴露。然而，大量研究证据表明，转录后调控机制——尤其是微小RNA（microRNAs，miRNAs）——在调控mRNA水平的动态变化中发挥着不可或缺的作用。那么，光线是否会直接影响miRNAs的功能？是否存在受光调控的miRNAs？若存在，它们靶向哪些类别的信使RNA（messenger RNA，mRNA）？为解答上述问题，我们对斑马鱼转录组中光诱导的表达变化开展了全面测序分析，涵盖小型非编码RNA与mRNA两类转录本。重要的是，我们鉴定出了多组受光调控的miRNAs，其众多调控靶点均为光诱导型mRNA，包括昼夜节律时钟基因以及参与氧化还原稳态的相关基因。我们随后聚焦于两种光响应性miRNA：miR-204-3-3p与miR-430a-3p，它们被预测可调控隐花色素基因（cryptochrome genes，cry1a与cry1b）的表达。荧光素酶报告基因实验验证了miR-204-3-3p与miR-430a-3p分别结合cry1a与cry1b的3′非翻译区（3′ untranslated region，3′UTR）的靶点序列。此外，使用这两种miRNA的模拟物与抑制剂进行处理，不仅显著改变了其靶基因的动态表达模式，还影响了其他核心时钟组分（clock1a、bmal1b、per1b、per2、per3）的表达，同时也改变了斑马鱼幼体的节律性运动活性。综上，我们对光响应性miRNAs的鉴定，揭示了光线对昼夜节律时钟进行多维度调控的全新复杂机制。