Suggestive evidence on the involvement of polypyrimidine-tract binding protein in regulating alternative splicing of MAP/microtubule affinity-regulating kinase 4 in glioma
Suggestive evidence on the involvement of polypyrimidine-tract binding protein in regulating alternative splicing of MAP/microtubule affinity-regulating kinase 4 in glioma
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DOI：
10.1016/j.canlet.2014.12.049
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发表时间：
2015-04-01
期刊：
影响因子：
9.7
通讯作者：
Larizza, L.
中科院分区：
文献类型：
--
作者：
Fontana, L.;Rovina, D.;Larizza, L.
MAP/微管亲和力调节激酶 4 (MARK4) 是一种丝氨酸-苏氨酸激酶，可磷酸化参与微管动力学调节的微管相关蛋白。 MARK4 以两种剪接亚型表达，其特征是外显子 16 的包含 (MARK4S) 或排除 (MARK4L)。中枢神经系统中的不同表达谱及其在神经胶质瘤中的不平衡分别表明 MARK4L 和 MARK4S 在细胞增殖和细胞分化中的作用。排除突变和转录缺陷后，我们假设剪接因子表达的改变可能是神经胶质瘤中 MARK4 表达失调的基础。生物信息学分析显示 MARK4 内含子 15 和 16 中有四个假定的多嘧啶束结合 (PTB) 蛋白结合位点。与正常大脑相比，神经胶质瘤组织和胶质母细胞瘤来源的癌症干细胞显示，PTB 显着过度表达，与 MARK4L mRNA 高表达相关。剪接小基因检测揭示了 MARK4 内含子 15 中存在功能性内含子剪接沉默子，但该区域中 PTB 结合位点的诱变并不影响小基因剪接，表明 PTB 可能与预测的剪接沉默子以外的剪接沉默子结合，并与其他预测的 PTB 位点协同作用。电泳迁移率变动分析与质谱联用证实了 PTB 与内含子 15 的多嘧啶束的结合，从而证实了其参与 MARK4 选择性剪接。这一发现与胶质瘤和胶质母细胞瘤衍生的癌症干细胞和分化后代中 PTB 过度表达的证据一起，共同指出 PTB 参与向 MARK4L 的转变，这与其在脑肿瘤中驱动致癌剪接的既定作用一致。 (C) 2014 Elsevier Ireland Ltd. 保留所有权利。
MAP/microtubule affinity-regulating kinase 4 (MARK4) is a serine-threonine kinase that phosphorylates microtubule-associated proteins taking part in the regulation of microtubule dynamics. MARK4 is expressed in two spliced isoforms characterized by inclusion (MARK4S) or exclusion (MARK4L) of exon 16. The distinct expression profiles in the central nervous system and their imbalance in gliomas point to roles of MARK4L and MARK4S in cell proliferation and cell differentiation, respectively. Having ruled out mutations and transcription defects, we hypothesized that alterations in the expression of splicing factors may underlie deregulated MARK4 expression in gliomas.Bioinformatic analysis revealed four putative polypyrimidine-tract binding (PTB) protein binding sites in MARK4 introns 15 and 16. Glioma tissues and glioblastoma-derived cancer stem cells showed, compared with normal brain, significant overexpression of PTB, correlated with high MARK4L mRNA expression. Splicing minigene assays revealed a functional intronic splicing silencer in MARK4 intron 15, but mutagenesis of the PTB binding site in this region did not affect minigene splicing, suggesting that PTB may bind to a splicing silencer other than the predicted one and synergistically acting with the other predicted PTB sites. Electrophoretic mobility shift assays coupled with mass spectrometry confirmed binding of PTB to the polypyrimidine tract of intron 15, and thus its involvement in MARK4 alternative splicing. This finding, along with evidence of PTB overexpression in gliomas and glioblastoma-derived cancer stem cells and differentiated progeny, merged in pointing out the involvement of PTB in the switch to MARK4L, consistent with its established role in driving oncogenic splicing in brain tumors. (C) 2014 Elsevier Ireland Ltd. All rights reserved.