Analysis of Traf3 alternative splicing during T cell activation

Genome-wide analyses of splicing patterns in resting and activated T cells have indicated a large number of genes to be alternatively spliced upon T cell activation. However, a functional role and the mechanism of splicing regulation has been established for very few of these alternative splicing events. In the present work we show that Traf3 alternative splicing is regulated upon activation of a T cell line as well as in primary T cells. To start analyzing the underlying mechanism we have cloned a minigene that recapitulates exon skipping upon T cell activation. Mutational analysis then enabled us to identify intronic regions that contribute to splicing regulation.

As Traf3 has been implicated in the regulation of NFkB activation, we have investigated the role of Traf3 alternative splicing in NFkB pathways. We present data that the Traf3 splicing switch observed upon T cell activation regulates activation of the non-canonical NFkB pathway. Consistently, a reporter gene under control of the non-canonical NFkB heterodimer was responsive to Morpholino-mediated manipulation of Traf3 isoform expression. In addition, expression of endogenous target genes of the non-canonical NFkB pathway was increased by altered Traf3 isoform expression. Our data thus suggest that Traf3 alternative splicing upon T cell activation controls activity of the non-canonical NFkB pathway which is critically involved in regulating immune function.