No abstract

Variations in ambient temperature present a unique obstacle to the timekeeping function of circadian clocks. Most biological reactions proceed with a temperature coefficient (Q 10 ) ∼ 2 or 3, so that with every 10 °C increase in temperature, the reaction rate approximately doubles or triples. Therefore, a temperature-dependent clock would run faster at high temperatures than at low temperatures, and would not be a reliable predictor of time of day. So it is not surprising that mechanisms have evolved to ensure that the length of the circadian period remains relatively constant over a wide range of temperatures, a phenomenon known as temperature compensation.In mammals, self-sustaining rhythms have been measured in the master circadian clock, located in the SCN, and in peripheral tissues in vitro (Yamazaki et al., 2000;Yoo et al., 2004). When separated from the entrainment of the SCN, each peripheral tissue expresses tissue-specific differences in circadian period and phase (Yoo et al., 2004). While previous studies have shown that the circadian period in the SCN, retina, and in fibroblast cell lines remains relatively constant across a range of temperatures, it is unknown whether mammalian peripheral clocks are temperature compensated (Tosini and Menaker, 1998;Ruby et al., 1999;Izumo et al., 2003;Tsuchiya et al., 2003).To examine the effect of temperature on circadian oscillations, we measured PERIOD2::LUCIFERASE (PER2::LUC) rhythms in explants of central and peripheral tissues from mPer2 Luc mice at 29, 31, 33, 35, and 37 °C. At temperatures ranging from 31 to 37 °C, robust rhythms of PER2::LUC were measured in the SCN, pituitary gland, cornea, adrenal gland, and lung. In the liver, the rhythm of PER2::LUC was robust at 37 °C, but damped within 2 cycles at all other temperatures examined. At 29 °C, only the pituitary consistently maintained a robust PER2::LUC rhythm.To determine if circadian clocks in mammalian central and peripheral tissues were temperature compensated, we calculated the average period of each tissue at various temperatures. The Q 10 of the SCN, pituitary gland, cornea, adrenal gland, and lung were variable, but all were close to 1, suggesting that these tissues were temperature compensated (Fig. 1). Because the PER2::LUC rhythm of liver explants was not robust at temperatures below 37 °C, the Q 10 of the liver could not be calculated. Since Per2 participates in the transcriptional/translational feedback loops that regulate the expression of other circadian genes, temperature-induced changes in the bioluminescent waveform of PER2::LUC expression could reflect variable processing of the components of the feedback loops. However, we found no differences in the waveforms of PER2::LUC expression between tissues cultured at 31 and 37 °C (Fig. 2). It is possible that the effects of temperature on the molecular mechanism of the clock are not reflected in rhythmic PER2::LUC expression and other circadian genes should also be assessed. Alternatively, our method may not be sensitive enough to detect tem...

We previously created two PER2::LUCIFERASE (PER2::LUC) circadian reporter knockin mice that differ only in the 3'-UTR region:, which retains the endogenous 3'-UTR and, where the endogenous 3'-UTR was replaced by an SV40 late poly(A) signal. To delineate the in vivo functions of 3'-UTR, we analyzed circadian rhythms of mice. Interestingly, mice displayed more than threefold stronger amplitude in bioluminescence rhythms than mice, and also exhibited lengthened free-running periods (∼24.0 h), greater phase delays following light pulse, and enhanced temperature compensation relative to Analysis of the 3'-UTR sequence revealed that miR-24, and to a lesser degree miR-30, suppressed PER2 protein translation, and the reversal of this inhibition in augmented PER2::LUC protein level and oscillatory amplitude. Interestingly, mRNA and protein oscillatory amplitude as well as CRY1 protein oscillation were increased in mice, suggesting rhythmic overexpression of PER2 enhances expression of and other core clock genes. Together, these studies provide important mechanistic insights into the regulatory roles of 3'-UTR, miR-24, and PER2 in expression and core clock function.

Level III-retrospective study, therapeutic.