Seasonal Adaptation of a Circadian Clock

描述(申请人提供)：在这项提案中，我们将使用果蝇作为一个模型系统，以了解昼夜节律(E24小时)系统如何适应环境条件的季节性变化，重点是温度相对较少的角色。一个主要的基础是基于我们的长期工作，该工作表明，在黑腹盘藻中，关键时钟成分周期(PER)中3‘端内含子(称为dmpi8)的温度依赖的剪接是一个重要的“温度传感器”，它调节每日觉醒-睡眠周期的分布，引起季节性适当的反应。例如，在温暖的日子里，dmpi8内含子的剪接效率低下，导致每个RNA水平的下降，这一事件延长了中午的午睡时间，并可能将炎热的中午时间与干燥相关的风险降至最低。最近的研究表明，多个次优剪接信号[即5‘和3’剪接位点(Ss)]是dmpi8剪接效率的温度敏感性的基础，并赋予了D.Blackogaster将中午午睡延长到下午中晚的能力，这可能有助于其适应温和气候，在那里温暖的白天通常伴随着较长的热期。事实上，在几种自然局限于非洲赤道地区的果蝇中，每3‘末端剪接效率的温度依赖变化和每日尾迹活动谱的调整都不存在，这些物种的温度和日长全年几乎没有波动。与我们的假设一致，这些非热响应物种在其每3‘末端的内含子上有很强的5’和3‘s。低温可能稳定剪接因子与次优剪接信号的相互作用，为热校准提供基础。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，以了解调控每个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觉醒-睡眠模式。新发现的在PER中调节dmpi8剪接的不同的自然多态将被描述。我们还将确定dmpi8剪接的影响是否优先从PER表达的早晨或晚上大脑起搏中心和/或最近描述的觉醒/睡眠神经元介导。这一分析应该为我们最近发现的dmpi8剪接调节白天睡眠提供进一步的见解，表明PER在调节觉醒-睡眠状态中扮演着新的非昼夜角色。通过使用各种自然种群和果蝇物种进行比较研究，这项提议提供了一个独特的机会，将控制复杂行为的基因表达和神经回路的研究与生态和进化影响结合起来。从更广泛的角度来看，我们的工作表明，在剪接信号水平上操作的自然选择在生命形式的热适应中发挥着重要作用，这为替代剪接程序和转录组调控带来了广泛的影响，我们将使用大规模RNA测序技术来探索这些问题。 与公共健康相关：像所有动物一样，人类每天的醒-睡周期由称为生物钟的特殊细胞网络控制。昼夜节律系统不仅与日常计时有关，而且还会引起季节性的适当反应。这项提议将研究生物钟如何对温度的季节性变化做出反应，这对生命形式适应温带气候至关重要。

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.