Mechanism of thermal entrainment of the circadian clock by newly identified post-transcriptional regulation

概要

Recent genome-wide transcriptome and proteome studies have revealed that mRNA expression levels only explain approximately 40% of the variation of protein product levels in cells, suggesting a potential importance of changeable protein production efficiency. The advent of sequencing technologies for evaluating RNA modifications led to the transformation of the concept on mRNA regulation, where mRNA is not an entity mainly controlled by copy number but is recognized to be a subject regulating protein expression through post-transcriptional modifications. Post-transcriptional regulation may thus serve as a platform for the regulation of flexible protein expression. Post- transcriptional regulatory element (PTRE), located in the untranslated region (UTR) of transcript, regulates its coding protein expression without affecting mRNA expression level. The molecular mechanisms underlying PTRE-dependent post-transcriptional regulation, however, are only limitedly understood, currently. Particularly, its physiological significance is completely unknown.

The 24-hour rotation of the Earth creates temporal changes in the environment, forcing the organisms to acquire the ability to adapt to recurrent, thus anticipatable environmental changes. A variety of physiological phenomena such as thermogenesis and sleep-wake cycles have been identified to exhibit daily fluctuation. Body temperature of thermostatic animals does not stay constant but displays a regular circadian fluctuation, which has an important physiological role in maintaining homeostasis of sleep and metabolism as well as entraining the peripheral circadian clocks in the body. Indeed, a subtle circadian fluctuation in body temperature within a physiologic range in vivo (35°C to 38.5°C in mice) has the ability to adjust or entrain the phase of the circadian clock in cultured cells. Although some of the heat or cold stress-related molecular regulators, such as the heat shock factor 1 and cold-inducible RNA-binding protein, are reported to participate in this entrainment mechanism, the precise mechanism(s) by which the physiological body temperature fluctuation affects the oscillation of the molecular clock in cells have remained unclear. One of the reasons for this unclarity is that most of previous studies have mainly focused on the contribution of transcription and paid less attention to post-transcriptional regulation in the control of the circadian clock.

In this research background, I found a specific PTRE sequence in the UTR of thermosensitive clock oscillating gene (Tsco) (Chapter 1). Importantly, a mild increase in temperature in a physiological range (warming temperature shift, hereafter WTS) led to increased Tsco protein expression, which is regulated at a post-transcriptional level in a PTRE-dependent manner (Chapter 2). To investigate the molecular mechanism underlying the temperature response of Tsco, I performed chemical library screening and revealed that blocking TRK35 abrogated WTS-dependent Tsco protein accumulation (Chapter 3). Finally, I showed that TRK35 and Tsco PTRE have a significant contribution to the establishment of the temperature entrainment of the circadian clock (Chapter 4).

Chapter 1: Discovery of Tsco PTRE in cells treated with a warm temperature shift
To investigate how WTS affects Tsco expression, I performed customized RNA-seq (CRNA-seq) and found increased recruitment of RNA-binding proteins (RBPs) to the UTR of Tsco mRNA in cultured mouse embryonic fibroblast (MEF) cells when a WTS (35°C to 38.5°C) was applied. In-depth sequence analysis revealed that the accumulation signal of RBPs on the UTR of Tsco transcript is located within a specific PTRE sequence. Importantly, this sequence is conserved among mammalian species, which include human and mouse.

Chapter 2: Temperature-dependent control of Tsco protein synthesis depends on PTRE. Physiological temperature changes might modulate Tsco expression. To test this, I performed immunoblot analysis using MEF cells and revealed that WTS induced greater expression of Tsco protein at its circadian rising phase, but not its circadian decreasing phase. Despite the change of Tsco protein expression by WTS, Tsco transcripts were nearly unaffected by the same treatment. WTS did not affect the Tsco protein stability. Next, I prepared mouse lung fibroblasts (MLF) from wild-type and Tsco PTRE mutant mice and found that WTS upregulated Tsco protein expression level in the wild-type cells but not in mutant cells. I further performed luciferase reporter assays using reporter constructs containing Tsco PTRE sequence and revealed that the Tsco PTRE sequence is sufficient for the temperature response of Tsco.

Chapter 3: Blocking temperature responsive kinase 35 abrogates WTS response of Tsco expression.
As a tool to identify an essential pathway for the temperature response of Tsco protein synthesis, I generated reporter knock-in cells in which a temperature insensitive luciferase gene was inserted in frame before the endogenous stop codon of Tsco, and using this tool, I performed chemical library screening. The screening revealed that WTS response of Tsco protein expression was attenuated in the presence of TRK35 inhibitors. Conversely, increased expression of Tsco protein was observed in the cells treated with a TRK35 pathway activator TPA. Very importantly, TPA failed to increase Tsco protein expression in Tsco PTRE mutant cells. These results indicate that WTS amplifies Tsco protein expression level through the TRK35-PTRE pathway.

Chapter 4: Thermal entrainment is mediated by TRK35-Tsco PTRE pathway.
I verified that temperature cycles that simulated mouse body temperature were able to enhance circadian rhythm sustainability in wild-type cells. However, the temperature cycle failed to maintain the rhythmicity in Tsco PTRE mutant cells. Inhibition of TRK35 also attenuated the temperature- cycle-dependent circadian sustainability of Tsco expression rhythm. Next, I examined the ability of temperature to synchronize or entrain the Tsco rhythm. The simulated mouse body temperature rhythm was capable of synchronizing the phase of Tsco expression in wild-type cells. In contrast, the temperature entrainment of Tsco was no longer observed in the presence of TRK35 inhibitor. These results demonstrate that the regulation of Tsco expression through TRK35-PTRE pathway is important for the thermal entrainment of the circadian clock.

Based on the results from Chapters 1–4, I have, for the first time, experimentally demonstrated the physiological contribution of post-transcriptional regulation of protein expression to temperature entrainment of the circadian clock by identifying the temperature-dependent response of the post- transcriptional regulatory element in the UTR of the thermosensitive clock gene.