An integrated transcriptomic-proteomic landscape of temperature controlled alternative splicing

选择性剪接（Alternative splicing， AS）通过外显子的不同组合，产生来自同一基因的多个转录本，在多种病理生理过程中发挥重要作用。由于新一代测序技术的快速发展，AS事件已经在不同生物体的RNA水平上得到了深入的表征。然而，相比之下，在蛋白质水平上的相应分析相当有限，主要是由于与测量多种蛋白质同种异构体相关的技术挑战。迄今为止，在RNA水平上检测到的选择性剪接转录物中只有一小部分得到了蛋白质水平证据的支持。因此，AS对蛋白质多样性的产生有多大的贡献在很大程度上尚不清楚。为了解决这个开放的基本问题，我们建议用转录组学、蛋白质组学和功能方法的独特组合来研究哺乳动物细胞中的AS。我们将重点关注体温依赖性AS作为我们的生物模型系统，因为(i)它与哺乳动物的昼夜节律相关，（ii）它有助于发烧诱导的免疫反应，（iii）它是进化保守的，（iv）它可以很容易地在组织培养细胞中进行研究。虽然我们之前的工作表明，温度控制的AS在细胞系和物种中广泛存在并保守，但到目前为止，我们主要研究的是RNA水平的变化。在这个项目中，我们将研究RNA水平上异构体表达的变化如何转化为蛋白质异构体的变化，以及这如何控制所产生的蛋白质的表达和功能。更具体地说，Chen实验室将结合先进的下一代基于测序的方法来获得一套完整的全长转录本，这些转录本以温度依赖的方式差异表达和/或翻译。Selbach实验室将采用已建立的蛋白质组学方法，并开发新技术，以单异构体分辨率量化蛋白质的稳态丰度、合成和降解率。广泛的转录组学和蛋白质组学数据将被整合，以解决基本的生物学问题，即mRNA同种异构体表达的改变在多大程度上导致了蛋白质组的复杂性。最后，海德实验室将进行功能实验，以评估温度依赖性AS的生物学后果。这里概述的建议依赖于我们的综合专业知识以及所涉及的pi之间的密切和长期合作，这两者都是成功的关键。我们将在一个令人兴奋的生物系统中提供迄今为止最深入的系统转录组学和蛋白质组学分析。我们期望获得关于AS基本原理的新见解以及与哺乳动物温度依赖生物学高度相关的结果。

Alternative splicing (AS) generates multiple transcripts from the same gene by different combinations of exons, and plays an important role in various patho-physiological processes. Thanks to the rapid development of next-generation sequencing technologies, AS events have been intensively characterized at the RNA level in different organisms. However, in comparison, the corresponding analysis at the protein level is rather limited, mainly due to the technical challenges associated with measuring multiple protein isoforms. Only a small proportion of the alternatively spliced transcripts detected at RNA level have thus far been supported with protein level evidence. Therefore, how much AS contributes to generate protein diversity is largely unclear. To address this open fundamental question, we propose to study AS in mammalian cells with a unique combination of transcriptomic, proteomic and functional approaches. We will focus on body temperature dependent AS as our biological model system because (i) it is relevant for the circadian rhythm in mammals, (ii) it contributes to fever-induced immune responses, (iii) it is evolutionarily conserved and (iv) it can be easily studied in tissue culture cells. While our previous work showed that temperature-controlled AS is widespread and conserved across cell lines and species, we have so-far mainly addressed changes at the RNA level. In this project, we will investigate how changes in isoform expression at the RNA level are translated into changes in protein isoforms and how this controls expression and function of the resulting proteins. More specifically, the Chen lab will use a combination of advanced next generation sequencing-based methods to obtain a comprehensive set of full-length transcripts that are differentially expressed and/or translated in a temperature dependent manner. The Selbach lab will employ established proteomic approaches and develop new technologies to quantify the steady-state abundance, synthesis and degradation rates of proteins at single isoform resolution. The extensive transcriptomic and proteomic data will be integrated to address the fundamental biological question, i.e., to which degree altered mRNA isoform expression contributes to proteome complexity. Finally, the Heyd lab will perform functional experiments to assess the biological consequences of temperature-dependent AS. The proposal outlined here relies on our combined expertise as well as the intensive and long standing collaborations between the PIs involved, both of which are essential for success. Together, we will provide the so far deepest systematic transcriptomic and proteomic analysis of AS in an exciting biological system. We expect to obtain both new insights on the fundamental principles of AS and results that are highly relevant for temperature-dependent biology in mammals.