Seasonal Adaptation of a Circadian Clock

描述（由申请人提供）：在本提案中，我们将使用果蝇作为模型系统来理解昼夜节律（E24小时）系统如何适应环境条件下的季节变化，重点是温度相对未表征的作用。一个主要的基础是基于我们长期的工作，表明在D. melanogaster中，来自关键时钟成分周期（per）的3'端内含子（称为dmpi8）的温度依赖性剪接是一个突出的“热传感器”，可以调节日常觉醒-睡眠周期的分布，引发季节性适当的反应。例如，在温暖的日子里，dmpi8内含子的剪接效率低下，导致每RNA水平下降，这一事件延长了午睡时间，并可能将炎热的正午时分干燥的风险降至最低。最近的进展表明，多个次优剪接信号（即5‘和3’剪接位点（ss））是dmpi8剪接效率的热敏性的基础，并赋予了D. melanogaster将午觉延长到下午中下旬的能力，可能有助于其适应温带气候，在温带气候中，温暖的日子通常与长时间的高温有关。事实上，在一些自然局限于非洲赤道地区的果蝇物种中，每3'端剪接效率的温度依赖变化和每日尾流活动曲线的调整是不存在的，在非洲赤道地区，温度和白昼长度全年波动很小。与我们的假设一致，这些非热响应的物种在它们的每3‘端内含子上有很强的5’和3‘ ’。低温可能稳定了拼接因子与次优拼接信号的相互作用，为热校准提供了基础。我们将采取多方面的实验策略，包括生化、分子、细胞培养和全动物方法，以了解调节每个3'端内含子剪接效率的顺式和反式作用因子，以及它们如何调节果蝇的清醒-睡眠特征。在per中新发现的调节dmpi8剪接差异的自然多态性，可能存在地理差异，将被表征。我们还将确定dmpi8剪接的影响是否优先由预先表达的早晨或晚上大脑起搏器中心和/或最近描述的觉醒/睡眠神经元介导。这一分析将为我们最近的发现提供进一步的见解，即dpi8剪接调节白天睡眠，这表明per在调节清醒-睡眠状态方面具有新的非昼夜节律作用。通过对各种自然种群和果蝇物种进行比较研究，本提案提供了一个独特的机会，将基因表达和神经回路控制复杂行为的研究与生态和进化意义结合起来。从更广泛的角度来看，我们的工作表明，在剪接信号水平上运行的自然选择在生命形式的热适应中起着重要作用，这对其他剪接程序和转录组调控产生了广泛的影响，我们将利用大规模rna测序技术探索这些问题。

DESCRIPTION (provided by applicant): In this proposal we will use Drosophila as a model system to understand how circadian (E24 hr) systems adapt to seasonal changes in environmental conditions, with an emphasis on the relatively uncharacterized role of temperature. A major foundation is based on our long-standing work showing that in D. melanogaster the temperature-dependent splicing of the 3'-terminal intron (called dmpi8) from the critical clock component period (per) is a prominent "thermosensor" that adjusts the distribution of daily wake-sleep cycles, eliciting seasonably appropriate responses. For example, on warm days splicing of the dmpi8 intron is inefficient leading to decreases in per RNA levels, events that prolong midday siesta and likely minimize the risks associated with desiccation during the hot midday hours. Recent progress indicates that multiple suboptimal splicing signals [i.e., 5' and 3' splice sites (ss)] are the basis for the thermosensitivity in the splicing efficiency of dmpi8, and endow D. melanogaster with the ability to prolong its midday siesta into the mid-to-late afternoon, presumably facilitating its adaptation to temperate climates where warm days are typically associated with extended periods of heat. Indeed, temperature-dependent changes in per 3'-terminal splicing efficiency and adjustments in daily wake-activity profiles are absent in several species of Drosophila that are naturally restricted to Afro- equatorial localities, wherein temperature and daylength undergo little fluctuation throughout the year. Consistent with our hypothesis, these non-thermal responsive species have strong 5' and 3'ss on their per 3'- terminal introns. Low temperatures likely stabilize the interaction of splicing factors with suboptimal splicing signals, providing a basis for thermal calibration. We will undertake a multi-faceted experimental strategy that includes biochemical, molecular, cell-culture and whole animal approaches to understand the cis- and trans- acting factors regulating the splicing efficiency of per 3'- terminal introns and how they modulate wake-sleep profiles in Drosophila. Newly identified natural polymorphisms in per that differentially regulate dmpi8 splicing and might vary geographically will be characterized. We will also determine whether the effects of dmpi8 splicing are preferentially mediated from the per-expressing morning or evening brain pacemaker centers and/or the more recently described arousal/sleep neurons. This analysis should provide further insights into our recent discovery that dmpi8 splicing regulates daytime sleep, suggesting novel non-circadian roles for per in modulating wake-sleep states. By undertaking comparative studies using a wide variety of natural populations and Drosophila species, this proposal offers a unique opportunity to integrate studies on gene expression and neural circuits controlling complex behaviors with ecological and evolutionary implications. On a broader perspective, our work suggests that natural selection operating at the level of splicing signals plays an important role in the thermal adaptation of life forms, raising broad implications for alternative splicing programs and transcriptome regulation, issues we will explore using massive-RNA sequencing technology. PUBLIC HEALTH RELEVANCE: Like all animals, human's exhibit daily wake-sleep cycles that are controlled by a network of specialized cells called circadian clocks. The circadian system is not only involved in daily timing but also eliciting seasonably appropriate responses. This proposal will investigate how circadian clocks respond to seasonal changes in temperature, critical for the adaptation of life forms to temperate climates.