Alternative Splicing

In the post genome era it has become evident that the number of protein coding genes cannot be the sole determinant of an organism’s proteome complexity (Roberts and Smith, 2002) and mechanisms that increase protein diversity posttranscriptionally have gained much attention. Alternative splicing is a regulated process that allows a certain exon to be either included or skipped from the final message leading to the production of several or many mature mRNAs from a single pre-mRNA. This makes alternative splicing one of the most abundant mechanisms to control gene expression at the posttranscriptional level (Maniatis and Tasic, 2002). Alternative splicing takes place at a steady state level, e.g. in a tissue specific way, but is also highly dynamic and allows the cell to quickly react to changing environments, e.g. during differentiation or activation (Chen and Manley, 2009; Wahl et al., 2009). As the resulting protein isoforms often act in a different or opposing manner, even small splicing changes can have a strong impact on the protein’s function (Lynch, 2004).
The development of splicing sensitive microarrays and deep sequencing technology has allowed analyses of alternative splicing on a genome wide scale, and led to the conclusion that as much as 95% of human multi-exon pre-mRNAs are alternatively spliced (Pan et al., 2008). 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 (Cooper et al., 2009). Further evidence that even a single alternative splicing event can have a fundamental impact on the cell’s functionality comes from the (very few) isoform specific mouse models that have been generated. Most of these mice show profound defects confirming the functional relevance of alternative splicing in vivo (Moroy and Heyd, 2007). The finding that splicing patterns differ markedly between different cell types, or different differentiation stages, led to the idea of alternative splicing as another source of protein diversity that acts independently of transcription. Consequently, some tissue specific splicing factors have been identified that regulate splicing patterns of several pre-mRNAs (e.g. Warzecha et al., 2009), comparable to cell type specific transcription factors and their target genes. However, in only few cases splicing regulation has been linked to a single protein; the outcome of alternative splicing rather represents the sum of many regulatory proteins acting on one exon finally leading to an in-or-out decision (Heyd and Lynch, 2009). Therefore, subtle changes in many splicing regulatory proteins may also represent a means of generating cell type specific splicing patterns, which might partly explain why so few tissue specific splicing factors have been identified thus far.
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 few data exist to answer questions such as ‘Can a changed splicing pattern change the identity of a cell?’ or ‘Is a terminally differentiated cell still functional when the splicing profile is reprogrammed to be that of one of its precursors?’.
We are addressing such questions by investigate the functional impact of alternative splicing during T cell activation in cell culture as well as the mechanisms of splicing regulation and the cis- and trans-acting factors involved.


Our main research aims are:

Aim 1: Investigate the functional relevance of alternative splicing in the JSL1 T cell line. In the past years the human JSL1 T cell line has been 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. By transfecting JSL1 cells with Morpholino oligomers individual splice sites will be blocked to manipulate isoform ratios. Functional consequences in resting and activated cells will then be assessed by measuring some of the main markers of T cell activation.
Aim 2: Use primary mouse T cells and mutant mice to investigate the impact of alternative splicing in vivo. First, alternatively spliced targets in mouse T cells will be identified by deep sequencing of different purified T cell populations and/or by testing confirmed targets from aim 1 using isoform specific RT-PCR. The isoform ratio of some genes will be manipulated with splice site blocking morpholino oligomers using described procedures, either transfection of purified T cells, or in vivo administration in mice, and functional consequences will be assessed as in aim 1. Based on these results, promising candidates will be selected to produce mice with an altered isoform ratio of that gene and the impact on T cell development and activation will be investigated.
Aim 3: Characterize the mechanism of splicing regulation for functionally important targets. While aims 1 and 2 should yield a functional characterization of alternative splicing events during T cell activation, aim 3 will focus on the mechanism behind that regulation. Minigene constructs will be designed to recapitulate the splicing events of some functionally important targets and mutational analysis will allow the identification of required cis-regulatory RNA sequences. Such RNA elements will then be used to perform binding studies that should lead to the identification of trans-acting factors binding to this RNA to regulate splicing. Such experiments will yield splicing regulatory proteins with distinct activities in resting versus activated T cells, and the underlying signaling pathways can be analyzed. In addition, such splicing factors could be tested for their ability to regulate more of the splicing events investigated in aims 1 and 2, to potentially identify proteins that have a larger impact in establishing a functionally significant pattern of alternative splicing in T cells.