A molecular thermometer connects body temperature with alternative splicing

在高等真核生物中，选择性剪接在提高基因组编码能力和动态调节蛋白质表达方面发挥着重要作用。激活诱导的选择性剪接对细胞外条件的变化做出反应，使选择性剪接成为一种潜在的机制来调节基因表达的日间依赖性变化。因此，选择性剪接被假设参与调控哺乳动物的生物钟，但功能证据才刚刚开始出现，调控细节尚不清楚。根据我们对剪接因子U2AF26的长期兴趣，我们发现U2AF26本身是交替剪接的。最近，我们可以证明U2AF26的选择性剪接是昼夜节律和光诱导的，特别是在小鼠的小脑中，并在分子时钟和对时差的适应中发挥基础作用。另一种U2AF26异构体中的移码允许翻译成3-UTR，从而产生与苍蝇生物钟的中心成分同源的蛋白质结构域。我们的数据导致了一个模型，在该模型中，光诱导的U2AF26选择性剪接减缓了适应过程，从而稳定了生物钟，使其不受光：暗条件的变化。在确定了哺乳动物系统中第一个具有重要功能的昼夜节律剪接开关后，我们现在的目标是描述将输入光(或昼夜时间)转换为输出剪接的信号模块。我们将首先表征顺式作用的RNA元件，这是前mRNA中的序列，对于调节这个功能重要的剪接开关是必要的和充分的(目标1)。然后，我们将鉴定与该序列结合的反式作用蛋白，以调节选择性剪接。此外，我们将分析调节这些蛋白质的昼夜节律活动的信号级联(目标2)。这些详细的分子分析将使我们能够在系统范围内识别共调控基因(目标3)。为此，我们将使用与顺式作用元件(目标1)的序列比较，击倒/RNA-Seq和反式作用因子的CLIP(目标2)的序列比较，并使用RNA-Seq鉴定小脑中昼夜节律和光诱导的外显子。总之，我们的分析将首次阐明昼夜节律和光诱导的选择性剪接的调控。这将对理解昼夜节律基因的表达和调控信号诱导的昼夜节律环境中的选择性剪接做出重大贡献。由于我们正在分析的拼接开关在功能上很重要，我们还将对外设时钟在时差条件下的时钟重置机制提供新的见解。合并时间生物学和选择性剪接领域将在这些令人兴奋的研究领域开辟新的方向，并将形成进一步跨学科工作和合作的基础。

In higher eukaryotes, alternative splicing plays a fundamental role in increasing the genomes coding capacity and in dynamically regulating protein expression. Activation-induced alternative splicing in response to changing extracellular conditions makes alternative splicing a potential mechanism to regulate day-time dependent changes in gene expression. Therefore, alternative splicing has been hypothesized to be involved in regulating the mammalian circadian clock, but functional evidence is only beginning to emerge and regulatory details are as of yet missing. Following our longstanding interest in the splicing factor U2AF26, we found U2AF26 itself to be alternatively spliced. More recently we could show U2AF26 alternative splicing to be circadian and light-inducible in particular in mouse cerebellum and to play a fundamental role in the molecular clockwork and adaptation to jetlag. A frameshift in the alternative U2AF26 isoform allows translation into the 3-UTR thus generating a protein domain with homology to a central component of the fly circadian clock. Our data lead to a model in which light-induced U2AF26 alternative splicing slows down the adaptation process thus stabilizing the circadian clock against changes in light:dark conditions. Having identified the first functionally important circadian splicing switch in a mammalian system we are now aiming to characterize the signaling module that translates the input light (or circadian time) into the output splicing. We will first characterize the cis-acting RNA-element, which is the sequence within the pre-mRNA, that is necessary and sufficient to regulate this functionally important splicing switch (aim 1). We will then identify trans-acting proteins that bind to this sequence in order to regulate alternative splicing. Furthermore, we will analyze signaling cascades that mediate circadian activity of such proteins (aim 2). These detailed molecular analyses will then allow us to identify coregulated genes on a system-wide scale (aim 3). To this end we will use sequence comparison with the cis-acting element (aim 1), knock-down/RNA-Seq and CLIP with the trans-acting factor (aim 2) and identification of circadian and light-inducible exons in cerebellum using RNA-Seq. Together, our analyses will shed first light on the regulation of circadian and light-inducible alternative splicing. This will be a major contribution towards understanding circadian gene expression and the regulation of signal-induced alternative splicing in circadian settings. As the splicing switch we are analyzing is functionally important, we will also provide new insights into the mechanism of clock-resetting under jetlag conditions in peripheral clocks. Merging the fields of chronobiology and alternative splicing will open new directions in these exciting research areas and will form the basis for further interdisciplinary work and collaborations.