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. The recent development of splicing sensitive microarrays and deep sequencing technologies has allowed analyses of alternative splicing on a genome wide scale, and led to the conclusion that as much as 90% of human multi-exon pre-mRNAs are alternatively spliced, many of them in a tissue-, differentiation- or activation-status dependent manner. 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 catastrophic consequences of its failure.

Even though the existence of cell type specific splicing patterns is well acknowledged, a fundamental question has not been addressed: the contribution of alternative splicing to the functionality and the identity of a cell. Very little data exist to address the question whether a changed splicing pattern can reprogram a differentiation process or whether a particular splicing pattern is required for the functionality of a differentiated cell. We are addressing such questions by investigating the functional impact of alternative splicing during activation and differentiation of mouse and human T cells as well as in the mouse brain. Some of our recent projects are described in more detail below.

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In the past years the human JSL1 T cell line has become an invaluable tool to investigate signal induced alternative splicing during T cell activation, as many aspects of splicing regulation observed in primary human T cells are recapitulated in this cell line. Microarray and deep sequencing approaches have revealed genes whose splicing pattern changes robustly upon activation, but functional implications have not been investigated. We are using a variety of techniques, among them transfection of Morpholino oligomers to selectively manipulate isoform ratios, to characterize functional consequences of selected splice events during T cell activation. In addition to Jsl1 cells we are using primary human and mouse T cells to confirm our findings in a more physiological setting.

One of the targets we are working on is the adaptor protein Traf3, which has been described as a negative regulator of the non-canonical (nc) NFkB pathway. We were able to show that T cell specific and activation-dependent Traf3 alternative splicing forms a Traf3 variant that, in contrast to the full length protein, activates the non-canonical NFkB pathway upon T cell activation. This signaling pathway induces the expression of several cytokines with a potential role in regulating T cell dependent adaptive immunity.

Effector molecules, such as cytokines, that are produced during T cell activation need to be secreted in order to be biologically active. Protein secretion during T cell activation is a highly regulated process, but previous research has almost exclusively focused on post-Golgi compartments. We have identified Sec16, a protein involved in an earlier step in protein secretion, to be alternatively spliced during T cell activation. This led to our interest in the COPII machinery and its regulation during T cell activation. We are using confocal microscopy, protein export assays as well as protein-protein interaction studies and FACS analyses to characterize the activity of different Sec16 isoforms in regulating the shape and functionality of the COPII machinery in naive and activated T cells.


With our work we could show that two essential processes during T cell activation, production and secretion of effector molecules, are controlled by alternative splicing. These results suggest that alternative splicing has a much stronger impact on the functionality of activated T cells than previously anticipated and we assume that our continued analyses will uncover more splice events involved in controlling different aspects of T cell biology.

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In addition to the functional studies described above, we are investigating mechanisms of splicing regulation during T cell activation. 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. As the splicing pattern of some of our targets appears to be regulated by histone modifications, we have also established ChIP in order to gain a deeper understanding of this regulatory layer.

Such analyses ultimately reveal splicing regulatory proteins with differential activity in resting versus activated T cells responsible for alternative splicing regulation. This leads to the more fundamental question how cell type specific splicing patterns are established. To address such questions we have started to use human B cell lines to compare splicing patterns and expression and/or activity of splicing regulatory proteins with those observed in Jsl1 cells.

To increase our ability of identifying trans-regulatory proteins for a particular splice event, we have established a targeted siRNA screen in mouse and human cells. By knocking down around 100 splicing regulatory proteins in different cellular contexts (for example resting and stimulated Jsl1 cells) and then comparing splicing of a particular target, we have successfully identified trans-regulatory proteins for targets we and other groups are interested in.

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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 finding 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, 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. We now expand this initial work and investigate the mechanism of circadian U2AF26 alternative splicing as well as other aspects of U2AF26 function.

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