Montelli Stefano, Mazzotta Gabriella, Vanin Stefano, Caccin Laura, Corrà Samantha, De Pittà Cristiano, Boothroyd Catharine, Green Edward W, Kyriacou Charalambos P, Costa Rodolfo
Department of Biology, University of Padova, Padova, Italy.
Laboratory of Genetics, The Rockefeller University, New York, NY, USA.
J Biol Rhythms. 2015 Jun;30(3):217-27. doi: 10.1177/0748730415583575.
Previous analysis of Drosophila circadian behavior under natural conditions has revealed a number of novel and unexpected features. Here we focus on the oscillations of per and tim mRNAs and their posttranscriptional regulation and observe significant differences in molecular cycling under laboratory and natural conditions. In particular, robust per mRNA cycling from fly heads is limited to the summers, whereas tim RNA cycling is observed throughout the year. When both transcripts do cycle, their phases are similar, except for the very warmest summer months. We also study the natural splicing profiles of per and tim transcripts and observe a clear relationship between temperature and splicing. In natural conditions, we confirm the relationship between accumulation of the per(spliced) variant, low temperature, and the onset of the evening component of locomotor activity, first described in laboratory conditions. Intriguingly, in the case of tim splicing, we detect the opposite relationship, with tim(spliced) expression increasing at higher temperatures. A first characterization of the 4 different TIM protein isoforms (resulting from the combination of the natural N-terminus length polymorphism and the C-terminus alternative splicing) using the 2-hybrid assay showed that the TIM(unspliced) isoforms have a stronger affinity for CRY, but not for PER, suggesting that the tim 3' splicing could have physiological significance, possibly in temperature entrainment and/or adaptation to seasonal environments.
先前对果蝇在自然条件下昼夜节律行为的分析揭示了许多新颖且意想不到的特征。在这里，我们聚焦于周期基因（per）和 timeless 基因（tim）信使核糖核酸（mRNAs）的振荡及其转录后调控，并观察到在实验室条件和自然条件下分子循环存在显著差异。特别是，从果蝇头部提取的周期基因信使核糖核酸的强劲循环仅限于夏季，而tim 核糖核酸的循环则全年可见。当两种转录本都进行循环时，除了在最炎热的夏季月份外，它们的相位相似。我们还研究了周期基因和tim 转录本的自然剪接图谱，并观察到温度与剪接之间存在明确的关系。在自然条件下，我们证实了在实验室条件下首次描述的周期基因（剪接后）变体的积累、低温与运动活动夜间成分开始之间的关系。有趣的是，在tim 剪接的情况下，我们检测到相反的关系，即tim（剪接后）的表达在较高温度下增加。使用双杂交试验对4种不同的TIM蛋白异构体（由天然N端长度多态性和C端可变剪接组合产生）进行的首次表征表明，TIM（未剪接）异构体对CRY具有更强的亲和力，但对PER则不然，这表明tim 3'剪接可能具有生理意义，可能在温度同步和/或对季节性环境的适应中发挥作用。
