Activation-dependent TRAF3 exon 8 alternative splicing is controlled by CELF2 and hnRNP C binding to an upstream intronic element

Cell-type specific and inducible alternative splicing has a fundamental impact on regulating gene expression and cellular function in a variety of settings including activation and differentiation. We have recently shown that activation-induced skipping of TRAF3 exon 8 activates non-canonical NFkB signaling upon T cell stimulation, but the regulatory basis for this splicing event remains unknown. Here we identify cis- and trans-regulatory elements rendering this splicing switch activation-dependent and cell-type specific. The cis-acting element is located 340-440 nucleotides upstream of the regulated exon and acts in a distance-dependent manner, as altering the location reduces its activity. An siRNA screen followed by X-link IPs and mutational analyses identified CELF2 and hnRNP C as trans-acting factors that directly bind the regulatory sequence and together mediate increased exon skipping in activated T cells. CELF2 expression levels correlate with TRAF3 exon skipping in several model systems, suggesting CELF2 to be the decisive factor with hnRNP C being necessary but not sufficient. These data suggest an interplay between CELF2 and hnRNP C as the mechanistic basis for activation-dependent alternative splicing of TRAF3 exon 8 and additional exons and uncover an intronic splicing silencer whose full activity depends on the precise location more than 300 nucleotides upstream of the regulated exon.