Molecular Chronobiology and regulation of gene expression in dinoflagellates

这个项目涉及甲藻生物钟系统的两个不同方面，(I)产生内源计时信号的时钟机制和(Ii)将这些内部计时信号转换为观察到的生化节律的输出机制。由于高通量测序的进步，我们现在可以在我们的模型生物体中解决这些问题，尽管完全缺乏正向或反向遗传工具。*真核模型系统中时钟机制的一个简化观点涉及一个耦合的转录/翻译反馈环(TTFL)，即时钟基因转录/翻译产生的蛋白质产物反馈并抑制自身的转录。在这个模型中，时钟基因的mRNA和蛋白质都是有节奏地表达的，蛋白质相对于RNA是滞后的。然而，我们最近的转录组图谱显示，Linguloddium缺乏任何可检测到的有节奏的RNA。我们在这里建议通过仔细分析我们的蛋白质和磷蛋白测序实验的数据来检验这一假设，即翻译/翻译后反馈环可能取代该生物体中的TTFL。*我们有三个项目，旨在研究Lingulodina的生物钟和生物节律的生化机制。首先，我们将进行核糖体分析，这是对核糖体保护的mRNA片段的深度测序，将使我们能够确定其合成速率在昼夜周期中变化的蛋白质的身份和相。我们已经知道了10种蛋白质的昼夜合成模式，这些将有助于验证这项技术。*第二，我们将通过评估细胞中每日周期中总蛋白和磷蛋白的水平来检查不同节奏的分子基础。我们预计，大多数节律都将以不同蛋白质的数量或活性为分子基础。我们将使用LC MS/MS测序来量化大约6000个未经修饰的蛋白质的水平，以寻找蛋白质数量的变化，并使用3000个磷酸蛋白质来评估蛋白质活性的潜在变化。我们预计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，我们希望解决mRNA有节奏翻译的潜在机制。我们对两种受调控的蛋白质特别感兴趣，每种蛋白质占总mRNA群体的~1%，因为我们预测，任何介导这些信息的翻译控制的因素也将相当丰富。我们将在昼夜周期内进行RNA-蛋白质相互作用组谱分析，这是一种通过LC-MS/MS蛋白质测序识别与mRNAs结合的蛋白质的技术。候选调控因子将与与上述测量的合成速率一致的信使核糖核酸库具有时间依赖性的关联。

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.*****