Does an ancient circadian clock control transcriptional rhythms using a non-transcriptional oscillator?

我们建议解开一个新发现的生物钟，它是所有生命形式共有的。人类的睡眠-觉醒周期是最熟悉的24小时节律，但事实上，这种“昼夜节律”几乎存在于所有生物体内。驱动这些节律的生物钟在所有生物体中都具有非常相似的特性。在动物、苍蝇、真菌、植物、古细菌和蓝藻中，它在人为恒定的环境中持续产生持续时间接近24小时的节律，并且在不同温度下其节律的持续时间异常稳定。大约从1995年开始，世界各地的实验室发现，所有这些生物体的生物钟机制都涉及基因调控网络。几个关键的“时钟基因”通过有节奏地关闭彼此的表达形成一个定时循环。令人惊讶的是，这些明显相似的时钟在每一组生物中依赖于完全不同的基因。生物学上的标准是，行为相似的生理过程也有相似的机制，都是从一个共同的祖先那里遗传下来的。时钟似乎有几个不同的起源，通过趋同进化获得了相似的行为。2005年，在蓝藻中发现了一种非遗传计时器，这一观点得到了加强。Kai振荡器有节奏地用磷酸盐分子修饰一个大的蛋白质，然后将它们移除。这似乎也是一种特殊的进化。其他生物的基因电路时钟通常包括一些由蛋白质磷酸化控制的，但它们的基因组缺乏蓝藻所需的Kai成分。我们最近的研究结果表明，这种模式在两个方面是错误的。在藻类和人类细胞中，至少有一部分生物钟机制不依赖于基因调控，而且生物钟的“非转录”部分似乎在所有生物体中都是共享的。其具体机制尚不清楚，我们建议在本项目中进行研究。首先，我们将跟进最近通过测试藻类中特定药物的作用而发现的线索，因为这些药物对细胞的生物化学有已知的影响。其次，我们将使用我们最近实施的一种技术方法来并行监测数百个蛋白质磷酸化事件，以便在基因调控受阻时发现任何仍然保持节律的事件。这些将代表非转录时钟的一部分，或者它控制的其他蛋白质（就像机械时钟的“指针”）。在简单的藻类中，这部分工作将更快更容易，因为它的蛋白质类型更少，而且因为我们已经找到了分别研究时钟每个部分的方法。我们将回顾大约30亿年的进化，以找到这个最早的时钟机制。我们会问，哪些过程的节奏至今仍在控制着。我们希望找出为什么它们如此重要，以至于非转录时钟被保存到现在。同样重要的是，要弄清楚非转录时钟是如何对研究人员迄今为止研究的节律（如基因节律和睡眠-觉醒周期）进行计时的。在我们知道是什么驱动非转录时钟之前，很难做到这一点，但这个项目应该提供我们需要的工具。当然，我们将测试我们在藻类中的结果是否也适用于其他生物，以证明这个古老的时钟是否仍然是所有生物中细胞寿命的计时。如果是这样，那么这个原始的细胞计时器可能是未来治疗睡眠障碍的关键，帮助其他藻类在阳光明媚的时候产生生物燃料，以及未来作物在不可预测的气候下在可预测的时间开花。

We propose to unwind a newly-discovered biological clock, that is shared by all forms of Life. The human sleep-wake cycle is the most familiar 24-hour rhythm, but in fact such 'circadian rhythms' are found in almost all living organisms. The circadian clock, which drives these rhythms, shares very similar properties in all organisms. In animals, flies, fungi, plants, archaea and cyanobacteria, it continues to generate rhythms close to 24h in duration in artificially constant environments, and its rhythms are unusually stable in duration at different temperatures. Since roughly 1995, laboratories across the world have found that the clockwork mechanism of all these organisms involves networks of gene regulation. A few key "clock genes" form a timing loop by rhythmically turning off each other's expression. Surprisingly, these overtly similar clocks depend on quite different genes in each group of organisms. The norm in biology has been that physiological processes that behave alike also share similar mechanisms, all inherited from a common ancestor. Clocks appeared to have several different origins, that gained similar behaviour through convergent evolution. This notion was reinforced when, in 2005, a non-genetic timer was discovered in cyanobacteria. The Kai oscillator rhythmically decorated a large protein with phosphate molecules, then removed them. This too seemed an idiosyncratic piece of evolution. The gene-circuit clocks in other organisms often included some control by protein phosphorylation, but their genomes lacked the Kai components that were required in cyanobacteria.Our recent results suggest that this paradigm is wrong on two counts. At least part of the clock mechanism in an alga and in human cells does not depend on gene regulation, and this 'non-transcriptional' part of the clock appears to be shared across all organisms. Its detailed mechanism is unknown, and we propose to study it in this project. Firstly, we will follow up leads that we have recently uncovered by testing the effects of specific drugs in the alga, because the drugs have known effects on the cell's biochemistry. Secondly, we will use a technological method that we recently implemented to monitor hundreds of protein phosphorylation events in parallel, in order to find any that still remain rhythmic when gene regulation is blocked. These will represent either parts of the non-transcriptional clock, or other proteins that it controls (like the 'hands' of a mechanical clock). This part of the work will be faster and easier in the simple alga, because it has fewer protein types, and because we have found ways to study each part of the clock separately.We will be looking back about 3 billion years in evolution, to find this earliest clock mechanism. We will ask which processes its rhythms still control today. We hope to find out why these were so important that the non-transcriptional clock has been preserved to the present. It is also important to find out how the non-transcriptional clock contributes to timing the rhythms that researchers have studied up to now, like the gene rhythms and the sleep-wake cycle. Until we know what drives the non-transcriptional clock, it will be difficult to do so, but this project should provide the tools we need. Of course, we will then test whether our results in the alga also hold in other organisms, to show whether this ancient clock still times the lives of cells in all of Biology. If so, then this original cellular timer could hold the key to future treatments for sleep disorders, to helping other algae produce biofuel while the sun shines, and to future crops that flower at predictable times in an unpredictable climate.