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In the post genome era, it has become evident that in higher eukaryotes mechanisms other than regulated transcription play a fundamental role in controlling gene expression. Alternative splicing multiplies the number of possible proteins generated from a single pre-mRNA and therefore is one of the most abundant mechanisms to control protein expression and function posttranscriptionally. Indeed, above 90% of human multi-exon pre-mRNAs are alternatively spliced, many of them in a tissue-, differentiation- or activation-status dependent manner. In addition, splicing patterns are controlled in a species-specific manner and the abundance of alternative splicing correlates with organism complexity, suggesting a role of alternative splicing in the formation of species-specific traits. The ever-growing number of human diseases associated with splicing defects confirms the fundamental impact of (alternative) splicing on generating a functional cell in vivo and demonstrates the severe consequences of its failure.

Even though the existence of cell type and species-specific splicing patterns is well acknowledged, two fundamental questions are only beginning to be addressed: how are particular splicing patterns established and what is their contribution to the functionality and the identity of a cell or a whole organism? We are addressing such questions by investigating the regulation and functional impact of alternative splicing during activation of mouse and human T cells as well as in the brain, for example in a time of day dependent manner. Some of our recent and ongoing work is described in more detail below.
 

Different ways of analyzing alternative splicing. The left panel shows a global analysis of tissue specific alternative splicing using RNA-Seq, represented as heat map of percent spliced in (PSI) values. The middle panel shows rhythmic U2AF26 alternative splicing in a cell culture model for circadian splicing regulation using (semi-)quantitative, radioactive RT-PCR. The right panel shows an in vitro splicing assay where addition of a recombinant protein controls alternative splicing.
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In recent work we were able to show that the splicing factor U2AF26 is highly expressed in activated T cells and mouse brain and is itself alternatively spliced. Based on this initial observation we found that U2AF26 isoform expression in the brain is strongly regulated with the circadian rhythm, with a particular splice variant being only present at certain Zeitgeber times. We could then show that this U2AF26 variant regulates turnover of another core clock component, Period1, thus establishing the first functionally important circadian splicing switch in a mammalian system. Furthermore, recently generated U2AF26 deficient mice show altered adaptation of the clockwork in response to jetlag, confirming the relevance of U2AF26 for the circadian clock in vivo (Preussner et al., 2014, Mol Cell).

Based on this work, we have addressed the mechanistic basis for circadian alternative splicing. Using cell culture as well as in vivo models we have identified a large group of exons whose splicing pattern responds fast and extremely sensitive to temperature changes in the physiological temperature range. As many of these exons show rhythmic splicing in vivo, we suggest that this ‘splicing based thermometer’ uses circadian changes in body temperature as input to control a concerted splicing change in a group of exons in functionally related genes. We are now addressing the functionality of some of these temperature-sensitive splicing events and are investigating the molecular events that directly act as temperature sensor.

In addition, we are using a primary cell culture system to address the role of other (pre-)mRNA processing events in controlling rhythmic accumulation of circadian output genes. 
 

U2AF26 alternative splicing in murine cells exposed to the indicated temperature for 2 hours. An almost perfect linear correlation is observed for the isoform lacking exons 6 and 7 and temperature.
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The transport of secretory proteins from the endoplasmic reticulum (ER) to the Golgi depends on COPII-coated vesicles. While the basic principles of the COPII machinery have been identified, it remains largely unknown how COPII transport is regulated to accommodate tissue- or activation-specific differences in cargo load and identity. To address this question we have used T cells as a model system that show strongly increased secretory cargo load upon activation. We have confirmed increased ER-export efficiency in activated T cells and then used RNA-Seq to uncover the molecular mechanism for this adaptation. While we do not find substantial changes in the abundance of mRNAs encoding for components of the early secretory pathway, we observe a strong change in alternative splicing of Sec16, a protein essential for COPII vesicle generation. Using Morpholino-mediated manipulation of alternative splicing as well as CRISPR/Cas9-mediated genome engineering we show that this splicing switch controls the number of ER exit sites and transport efficiency. Our work provides the first connection between the COPII pathway and alternative splicing thus adding a new regulatory layer to protein secretion and its adaptation to changing cellular environments. As a mechanistic basis, we suggest the C-terminal Sec16 domain to be a splicing-controlled protein-interaction platform, with individual isoforms showing differential ability to recruit COPII components (Wilhelmi et al., 2016, Nat Comm). We are now generating isoform-specific Sec16 knock-out mice to correlate Sec16 alternative splicing with in vivo phenotypes. Based on this first example we are also analyzing a broader impact of alternative splicing in controlling the functionality of the early secretory pathway.
 

Model for the role of Sec16 alternative splicing in regulating ER-export during T cell activation (Wilhelmi et al., 2016, Nat Comm). Sec16 alternative splicing is differentially regulated in different cell types, suggesting an involvement in controlling protein secretion in a tissue-specific manner. 
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In addition to functional studies, we are investigating mechanisms that lead to distinct splicing patterns in different cell types, as a function of external stimuli or in a species-specific manner. Using minigene analyses and RNA-protein interaction methods such as X-link, pull-down and CLIP, we are aiming to characterize the cis- and trans-acting elements as well as signaling cascades that mediate splicing regulation of functionally important target genes. To be able to identify trans-regulatory proteins for a particular splicing event without analyzing cis-acting elements beforehand, we have established a targeted siRNA screen in mouse and human cells. We have acquired siRNA libraries that target around 200 (human) or 100 (mouse) splicing regulatory proteins and have successfully used these libraries in different cellular backgrounds to identify trans-acting factors that control alternative splicing of exons we and other groups are interested in (e.g. Meiniger et al., 2016, Nat Comm; Schultz et al., 2016, MCB).
 

Identification of Celf2 as a trans-acting factor that controls Traf3 alternative splicing upon T cell activation using an siRNA screen (Schultz et al., 2016, MCB).
 

As several of our target exons appear to be regulated in a species-specific manner, we have started to analyze alternative splicing patterns of orthologous exons in different species more systematically. For some targets we use minigene assays to investigate how species-specific splicing patterns are established. These analyses form the basis to investigate the functionality of species-specific alternative splicing and a potential role in defining species-specific traits.   

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