A molecular thermometer connects body temperature with alternative splicing

分子温度计通过选择性剪接连接体温

• 批准号: 270986915
• 负责人: Professor Dr. Florian Heyd
• 金额: --
• 依托单位: Arbeitsgruppe Heyd - RNA Biochemistry
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2015
• 资助国家: 德国
• 起止时间: 2014-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/270986915?language=en
• 关键词: molecular; thermometer; connects; body; temperature

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.

在高等真核生物中，选择性剪接在增加基因组编码能力和动态调节蛋白质表达方面起着重要作用。激活诱导的选择性剪接响应细胞外条件的变化，使选择性剪接的一个潜在的机制，以调节白天依赖性的基因表达的变化。因此，选择性剪接被假设参与调节哺乳动物的昼夜节律钟，但功能性证据才刚刚开始出现，而调控细节尚未找到。根据我们对剪接因子U2 AF 26的长期兴趣，我们发现U2 AF 26本身是选择性剪接的。最近，我们可以证明U2 AF 26选择性剪接是昼夜节律和光诱导的，特别是在小鼠小脑中，并在分子时钟和适应时差中发挥重要作用。替代U2 AF 26亚型中的移码允许翻译成3-UTR，从而产生与苍蝇昼夜节律钟的核心成分具有同源性的蛋白质结构域。我们的数据导致了一个模型，其中光诱导的U2 AF 26选择性剪接减缓了适应过程，从而稳定了昼夜节律钟，以对抗光暗条件下的变化。已经确定了哺乳动物系统中第一个功能上重要的昼夜节律剪接开关，我们现在的目标是表征将输入光（或昼夜节律时间）转化为输出剪接的信号传导模块。我们将首先表征顺式作用RNA元件，其是前体mRNA内的序列，其对于调节这种功能上重要的剪接开关是必要且足够的（目的1）。然后，我们将确定反式作用蛋白结合到这个序列，以调节选择性剪接。此外，我们将分析信号级联介导的昼夜节律活动的蛋白质（目的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节基因（目标3）。为此，我们将使用顺式作用元件的序列比较（目的1），敲除/RNA-Seq和CLIP与反式作用因子（目的2），并使用RNA-Seq鉴定小脑中的昼夜节律和光诱导外显子。我们的分析将共同揭示昼夜节律和光诱导选择性剪接的调节。这将是对理解昼夜节律基因表达和调节信号诱导的选择性剪接在昼夜节律设置的重大贡献。由于我们分析的拼接开关在功能上很重要，我们也将提供新的见解时钟重置机制下时差条件下的外围时钟。将时间生物学和选择性剪接领域合并将在这些令人兴奋的研究领域开辟新的方向，并将为进一步的跨学科工作和合作奠定基础。