An integrated transcriptomic-proteomic landscape of temperature controlled alternative splicing

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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.