Schultz Astrid-Solveig, Preussner Marco, Bunse Mario, Karni Rotem, Heyd Florian
Freie Universität Berlin, Institute of Chemistry and Biochemistry, Berlin, Germany.
Max Delbrück Center for Molecular Medicine, Berlin, Germany.
Mol Cell Biol. 2017 Mar 17;37(7). doi: 10.1128/MCB.00488-16. Print 2017 Apr 1.
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 noncanonical NF-κB signaling upon T cell stimulation, but the regulatory basis for this splicing event remains unknown. Here we identify - and -regulatory elements rendering this splicing switch activation dependent and cell type specific. The -acting element is located 340 to 440 nucleotides upstream of the regulated exon and acts in a distance-dependent manner, since altering the location reduces its activity. A small interfering RNA screen, followed by cross-link immunoprecipitation and mutational analyses, identified CELF2 and hnRNP C as -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 that CELF2 is 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.
细胞类型特异性和可诱导的可变剪接在包括激活和分化在内的多种情况下，对调节基因表达和细胞功能具有根本性影响。我们最近发现，激活诱导的TRAF3外显子8跳跃在T细胞刺激时激活非经典NF-κB信号，但这种剪接事件的调控基础仍然未知。在这里，我们鉴定了使这种剪接开关具有激活依赖性和细胞类型特异性的顺式作用元件和反式作用元件。顺式作用元件位于受调控外显子上游340至440个核苷酸处，并以距离依赖性方式起作用，因为改变其位置会降低其活性。通过小干扰RNA筛选，随后进行交联免疫沉淀和突变分析，鉴定出CELF2和hnRNP C作为反式作用因子，它们直接结合调控序列，并共同介导激活的T细胞中外显子跳跃增加。在几个模型系统中，CELF2表达水平与TRAF3外显子跳跃相关，这表明CELF2是决定性因素，而hnRNP C是必要但不充分的因素。这些数据表明CELF2和hnRNP C之间的相互作用是TRAF3外显子8和其他外显子激活依赖性可变剪接的机制基础，并揭示了一个内含子剪接沉默子，其完整活性更多地取决于受调控外显子上游300多个核苷酸的精确位置。
