Molecular Chronobiology and regulation of gene expression in dinoflagellates

甲藻的分子时间生物学和基因表达调控

• 批准号: RGPIN-2018-04220
• 负责人: Morse, David
• 金额: $ 3.06万
• 依托单位: Université de Montréal
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=765224
• 关键词: Molecular; Chronobiology; regulation; gene; expression

This project addresses two different aspects of the circadian system in the dinoflagellate Lingulodinium, (i) the mechanism of the clock that generates endogenous timing cues and (ii) the output mechanism whereby these internal timing cues are converted into the observed biochemical rhythms. Due to the advances in high throughput sequencing, we can now address these issues in our model organism despite a total lack of forward or reverse genetic tools. A simplified view of the clock mechanism in eukaryotic model systems involves a coupled transcription/translation feedback loop (TTFL), whereby the protein product produced by transcription/translation of a clock gene feeds back and inhibits its own transcription. In this model, both clock gene mRNA and protein are rhythmically expressed with protein phase-lagged with respect to RNA. However, our recent transcriptome profiling has shown Lingulodinium lacks any detectable rhythmic RNAs. We propose here to test the hypothesis that a translation/post-translation feedback loop may replace the TTFL in this organism by careful analysis of the data from our protein and phosphoprotein sequencing experiments. We have three projects designed to address the biochemical mechanisms of the clock and the biological rhythms in Lingulodinium. First, we will perform ribosome profiling, a deep sequencing of ribosome-protected mRNA fragments that will allow us to determine the identity and phase of proteins whose synthesis rate varies over the circadian cycle. We already know the circadian synthesis pattern of ten proteins, and these will serve to validate the technique. Second, we will examine the molecular basis of different rhythms by assessing the levels of total protein and phosphoprotein in the cells over the daily cycle. We anticipate that most of the rhythms will have a molecular basis in the amount or activity of different proteins. We will use LC MS/MS sequencing to quantitate levels of roughly 6000 unmodified proteins to look for changes in protein amounts and 3000 phosphoproteins to assess potential changes in protein activities. We anticipate that changes in protein synthesis rates will correlate with any changes in total protein levels.Lastly, we wish to address the mechanisms underlying rhythmic translation of mRNA. We are especially interested in two regulated proteins, each representing ~1% of the total mRNA population, since we predict that any factors mediating the translational control of these messages will also be quite abundant. We will perform RNA-protein interactome profiling over the circadian cycle, a technique that identifies the proteins bound to mRNAs by LC-MS/MS protein sequencing. Candidate regulatory factors will have a time dependent association with the mRNA pool that agrees with the synthesis rates measured above.

该项目解决了两个不同方面的昼夜节律系统的甲藻Lingulodinium，（i）的时钟机制，产生内源性的时间线索和（ii）输出机制，这些内部的时间线索被转换成观察到的生化节奏。由于高通量测序的进步，我们现在可以在我们的模式生物中解决这些问题，尽管完全缺乏正向或反向遗传工具。真核生物模型系统中的时钟机制的简化视图涉及耦合的转录/翻译反馈环（TTFL），由此由时钟基因的转录/翻译产生的蛋白质产物反馈并抑制其自身的转录。在该模型中，时钟基因mRNA和蛋白质都有节律地表达，蛋白质相对于RNA具有相位滞后。然而，我们最近的转录组分析表明Lingulodinium缺乏任何可检测的节奏RNA。我们建议在这里测试的假设，翻译/翻译后反馈回路可能会取代TTFL在这种生物体的仔细分析的数据，从我们的蛋白质和磷蛋白测序实验。我们有三个项目旨在解决生物钟的生化机制和Lingulodinium的生物节律。首先，我们将进行核糖体分析，对核糖体保护的mRNA片段进行深度测序，这将使我们能够确定蛋白质的身份和相位，这些蛋白质的合成速率在昼夜节律周期中变化。我们已经知道10种蛋白质的昼夜合成模式，这些将有助于验证该技术。其次，我们将通过评估细胞中总蛋白和磷蛋白在日常周期中的水平来研究不同节律的分子基础。我们预计，大多数的节奏将有不同蛋白质的数量或活动的分子基础。我们将使用LC MS/MS测序来定量大约6000种未修饰蛋白质的水平，以寻找蛋白质量的变化，并使用3000种磷蛋白来评估蛋白质活性的潜在变化。我们预期蛋白质合成速率的变化将与总蛋白质水平的任何变化相关。最后，我们希望阐明mRNA节律性翻译的机制。我们特别感兴趣的是两种受调节的蛋白质，每种蛋白质占总mRNA群体的约1%，因为我们预测，任何介导这些信息翻译控制的因子也将是相当丰富的。我们将在昼夜节律周期中进行RNA-蛋白质相互作用组分析，这是一种通过LC-MS/MS蛋白质测序鉴定与mRNA结合的蛋白质的技术。候选调节因子将与mRNA库具有时间依赖性关联，这与上面测量的合成速率一致。