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多个转录本，在多种病理生理过程中发挥重要作用。由于下一代测序技术的快速发展，AS事件已在不同生物体的RNA水平上得到了广泛的表征。然而，相比之下，蛋白质水平的相应分析相当有限，主要是由于与测量多种蛋白质亚型相关的技术挑战。到目前为止，只有一小部分在RNA水平检测到的选择性剪接转录本得到了蛋白质水平证据的支持。因此，AS在多大程度上有助于产生蛋白质多样性在很大程度上是不清楚的。为了解决这个开放的基本问题，我们建议研究AS在哺乳动物细胞中的转录组学，蛋白质组学和功能的方法的独特组合。我们将专注于体温依赖性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）它有助于发热诱导的免疫反应，（iii）它在进化上是保守的，（iv）它可以很容易地在组织培养细胞中进行研究。虽然我们以前的工作表明，温度控制的AS在细胞系和物种中是广泛和保守的，但到目前为止，我们主要关注RNA水平的变化。在这个项目中，我们将研究如何在RNA水平上异构体表达的变化被翻译成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实验室将使用先进的下一代测序方法的组合来获得一套完整的全长转录本，这些转录本以温度依赖性方式差异表达和/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，以单一异构体分辨率量化蛋白质的稳态丰度、合成和降解速率。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，在何种程度上改变的mRNA同种型表达有助于蛋白质组的复杂性。最后，Heyd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之间的深入和长期合作，这两者对于成功至关重要。我们将共同提供迄今为止最深入的系统性转录组学和蛋白质组学分析AS在一个令人兴奋的生物系统。我们希望获得新的见解的基本原则和结果，是高度相关的哺乳动物的温度依赖性生物学。

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.