Mechanisms and function of alternative splicing in the plant circadian clock

昼夜节律在自然界中无处不在：大多数生物体在生物过程、生长或行为中表现出强大的日常节律。我们最熟悉的节奏是我们的睡眠/觉醒周期，它被设定为当地时间，当穿越时区时，会产生时差。节律是由位于每个活细胞中的“生物钟”驱动的，即使在没有外部信号的情况下，生物钟也会继续运行。这些生物装置使生物体能够预测可预测的环境变化，如有规律的昼夜转换，并相应地调整其行为。生物钟由分子电路组成，形成反馈回路，产生时钟组件的振荡行为。通过与周期中不同的分子通路连接，生物钟能够调节一天中关键通路（如代谢、信号传导、生长和发育）的时间。生物钟故障会给生物体带来巨大的适应性成本；在人类中，它会导致几种疾病状态。生物钟可以在一定的温度范围内工作。这对于暴露在不断变化的环境中的植物尤其重要。化学反应速率对温度变化非常敏感，但面对每天的温度变化和季节变化的天气模式，生物钟保持不变。然而，如何实现这一目标尚不清楚。因此，本项目的一个关键目标是了解在温度变化中缓冲植物时钟的分子过程。这一点很重要，因为维持生物钟功能对最佳光合作用、生长和繁殖时间至关重要——这些因素影响种子丰度（作物的产量）。当基因表达时，DNA序列首先被复制成RNA（转录），RNA被加工，然后它指导相应蛋白质的合成（翻译）。在这个项目中，我们专注于破译转录后RNA加工（选择性剪接：AS）在模式植物拟南芥中温度依赖性时钟功能中的作用。AS从同一基因中产生不同的转录本，从而调节转录本和蛋白质的水平和功能。我们已经证明AS在控制时钟基因表达方面是重要的。我们将确定时钟输入基因中AS的程度，低温下如何影响AS以及它们对时钟基因表达/AS的影响。植物在整个昼夜循环中经历持续和可变的温度变化-我们将研究温度变化的程度和时间，这能够引发温度依赖的AS响应。光对生物钟也有重要的影响，我们将确定哪些AS事件对光强度变化有反应，以及它们是否与依赖温度的事件不同。一个主要的问题是不同时钟基因中不同类型的AS是如何被调节的，以及这种机制如何有助于昼夜节律时钟的温度缓冲。我们将使用RNA测序来评估冷却过程中的AS事件。共表达和共剪接网络分析将识别表达/剪接谱与核心时钟和时钟相关基因相关的基因，以识别推定的调控基因。我们还将研究这个过程中的自然遗传变异，以及这如何有助于我们的理解。AS在调控时钟基因表达中的作用与作物植株和产量密切相关。为了利用拟南芥研究的知识和方法，我们已经开始研究马铃薯和大麦中的AS控制，开始向应用转化。我们预计这项工作将为控制表型（如大麦的早熟和马铃薯的内休眠）提供新的见解，并最终导致产生新的基因型的策略，这些基因型增加了对温度和其他可能影响植物的胁迫的鲁棒性。

Circadian rhythms are ubiquitous in nature: most organisms exhibit robust daily rhythms in biological processes, growth or behaviour. The most familiar rhythm to us is our sleep/wake cycle which is set to local time and gives rise to jet lag when time-zones are traversed. Rhythms are driven by 'circadian clocks' located in every living cell that continue to run even in the absence of external signals. These biological devices allow an organism to anticipate predictable environmental changes such as the regular day to night transitions and to adjust its behaviour accordingly. Circadian clocks comprise molecular circuits organised into feedback loops that generate the oscillatory behaviour of clock components. By linking to different molecular pathways through the cycle, the clock is able to regulate the timing of key pathways (e.g. metabolic, signalling, growth and development) through the day. Clockwork failure can have a large fitness costs to the organism; in humans it contributes to several disease states.Circadian clocks can operate over a temperature range. This is particularly important for plants that are exposed to the ever-changing environment. Chemical reaction rates are very sensitive to temperature change, yet the clock remains invariant in the face of daily temperature changes and a range of weather patterns through the seasons. Yet how this is achieved is not known. A key objective for this project is therefore to understand molecular processes that buffer the plant clock from temperature changes. This is important as maintaining clock function is essential for optimal photosynthesis, growth, and the timing of reproduction - factors that influence seed abundance (a yield output for crop plants). When genes are expressed the DNA sequence is first copied into RNA (transcription), the RNA is processed and then it directs synthesis of the corresponding protein (translation). In this project we focus on deciphering the role of post-transcriptional RNA processing (alternative splicing: AS) in temperature-dependent clock function in the model plant Arabidopsis. AS generates different transcripts from the same gene and thereby can modulate transcript and protein levels and functions. We have shown that AS is important in controlling clock gene expression. We will establish the extent of AS in clock input genes, how this is affected at low temperature and their influence expression/AS of clock genes. Plants experience continued and variable temperature changes throughout the day/night cycle - we will investigate the degree and timing of temperature change which is able to elicit a temperature-dependent AS response. Light also has a major influence on the clock and we will identify which AS events respond to light intensity changes and whether they are different from temperature-dependent events.A major question is how the different types of AS in different clock genes are regulated and how this mechanism contributes to temperature buffering of the circadian clockwork. We will use RNA sequencing to assess AS events during cooling. Co-expression and co-splicing network analysis will identify genes whose expression/splicing profiles correlate with those of the core clock and clock-associated genes to identify putative regulatory genes. We will also examine the natural genetic variation in this process and how this might aid our understanding. The role of AS in regulation of clock gene expression is highly relevant to crop plants and yield. To utilise the knowledge and approaches from our Arabidopsis research, we have started to examine AS control in potato and barley to begin the translation toward application. We anticipate that this work will provide new insights into what controls phenotypes such as earliness in barley and endodormancy in potato and ultimately lead to strategies for the generation of new genotypes that have increased robustness to temperature and other stresses that can affect plants.