Mechanisms of Circadian Clock Control of mRNA Translation

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Project Summary The circadian clock is an evolutionarily conserved time-keeping mechanism that, through the regulation of rhythmic gene expression, coordinates the physiology of an organism with daily environmental cycles. Because nearly all aspects of human physiology and behavior are linked to the clock, clock dysfunction is associated with a wide range of diseases, including sleep disorders, cardiovascular disease, metabolic syndrome, and cancer. In addition, the clock controls the efficacy and toxicity of many drugs. Therefore, identifying what genes and proteins are regulated by the clock, and determining the mechanisms for this regulation, are key to understanding clock-associated diseases and rhythmic drug metabolism. While the primary focus of research on circadian control of gene expression has been at the transcriptional level, recent evidence supports a role for the clock in regulating posttranscriptional mechanisms. How the clock regulates mRNA translation is poorly understood. We found that the Neurospora crassa circadian clock controls the phosphorylation of two highly conserved central regulators of mRNA translation, eukaryotic elongation factor 2 (eEF2), and eukaryotic initiation factor 2α (eIF2α). Using high throughput RNA-seq and ribosome profiling in wild type cells, and cells that are defective in rhythmic activity of the translation factors, we found that clock regulation of translation factor activity affects translation of specific mRNAs, rather than acting globally to regulate translation of all mRNAs. During the next 5 years, we will leverage our expertise and tools to determine the mechanisms for this specificity. In an exciting breakthrough, we found that the clock controls the composition of cytoplasmic ribosomes, whereby certain ribosomal proteins cycle in abundance in ribosomes. These data challenge the paradigm that all cytoplasmic ribosomes are the same, and instead suggest the existence of heterologous ribosomes with distinct functions. We will capitalize on these findings to test the hypothesis that changes in ribosome protein composition, modification, and/or interactions with accessory proteins occur temporally under control of the clock. Elucidating the clock-regulated ribosome modification mechanism may also lead to insights into unexpected ways that signals other than the clock might postranscriptionally regulate protein expression.