Molecular Chronobiology and regulation of gene expression in dinoflagellates

点击翻译按钮获取中文摘要

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.*****