Melton Alexis A, Jackson Jason, Wang Jiarong, Lynch Kristen W
Department of Biochemistry, UT Southwestern Medical Center, Dallas, TX 75390-9038, USA.
Mol Cell Biol. 2007 Oct;27(19):6972-84. doi: 10.1128/MCB.00419-07. Epub 2007 Jul 30.
Cells can regulate their protein repertoire in response to extracellular stimuli via alternative splicing; however, the mechanisms controlling this process are poorly understood. The CD45 gene undergoes alternative splicing in response to T-cell activation to regulate T-cell function. The ESS1 splicing silencer in CD45 exon 4 confers basal exon skipping in resting T cells through the activity of hnRNP L and confers activation-induced exon skipping in T cells via previously unknown mechanisms. Here we have developed an in vitro splicing assay that recapitulates the signal-induced alternative splicing of CD45 and demonstrate that cellular stimulation leads to two changes to the ESS1-bound splicing regulatory complex. Activation-induced posttranslational modification of hnRNP L correlates with a modest increase in the protein's repressive activity. More importantly, the splicing factor PSF is recruited to the ESS1 complex in an activation-dependent manner and accounts for the majority of the signal-regulated ESS1 activity. The associations of hnRNP L and PSF with the ESS1 complex are largely independent of each other, but together these proteins account for the total signal-regulated change in CD45 splicing observed in vitro and in vivo. Such a combinatorial effect on splicing allows for precise regulation of signal-induced alternative splicing.
细胞可通过可变剪接响应细胞外刺激来调节其蛋白质组；然而，控制这一过程的机制仍知之甚少。CD45基因在T细胞激活时会发生可变剪接，以调节T细胞功能。CD45外显子4中的ESS1剪接沉默子通过hnRNP L的活性在静息T细胞中导致基础外显子跳跃，并通过此前未知的机制在T细胞中导致激活诱导的外显子跳跃。在此，我们开发了一种体外剪接测定法，该方法概括了CD45的信号诱导可变剪接，并证明细胞刺激会导致与ESS1结合的剪接调节复合物发生两种变化。激活诱导的hnRNP L的翻译后修饰与该蛋白抑制活性的适度增加相关。更重要的是，剪接因子PSF以激活依赖的方式被招募到ESS1复合物中，并占信号调节的ESS1活性的大部分。hnRNP L和PSF与ESS1复合物的结合在很大程度上相互独立，但这些蛋白质共同构成了体外和体内观察到的CD45剪接中信号调节的总变化。这种对剪接的组合效应允许对信号诱导的可变剪接进行精确调节。
