Tarn Woan-Yuh, Kuo Hung-Che, Yu Hsin-I, Liu Shin-Wu, Tseng Ching-Tzu, Dhananjaya Dodda, Hung Kuan-Yang, Tu Chi-Chiang, Chang Shuo-Hsiu, Huang Guo-Jen, Chiu Ing-Ming
Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan
Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan.
Mol Biol Cell. 2016 May 15;27(10):1676-83. doi: 10.1091/mbc.E15-11-0798. Epub 2016 Mar 23.
RBM4 participates in cell differentiation by regulating tissue-specific alternative pre-mRNA splicing. RBM4 also has been implicated in neurogenesis in the mouse embryonic brain. Using mouse embryonal carcinoma P19 cells as a neural differentiation model, we observed a temporal correlation between RBM4 expression and a change in splicing isoforms of Numb, a cell-fate determination gene. Knockdown of RBM4 affected the inclusion/exclusion of exons 3 and 9 of Numb in P19 cells. RBM4-deficient embryonic mouse brain also exhibited aberrant splicing of Numb pre-mRNA. Using a splicing reporter minigene assay, we demonstrated that RBM4 promoted exon 3 inclusion and exon 9 exclusion. Moreover, we found that RBM4 depletion reduced the expression of the proneural gene Mash1, and such reduction was reversed by an RBM4-induced Numb isoform containing exon 3 but lacking exon 9. Accordingly, induction of ectopic RBM4 expression in neuronal progenitor cells increased Mash1 expression and promoted cell differentiation. Finally, we found that RBM4 was also essential for neurite outgrowth from cortical neurons in vitro. Neurite outgrowth defects of RBM4-depleted neurons were rescued by RBM4-induced exon 9-lacking Numb isoforms. Therefore our findings indicate that RBM4 modulates exon selection of Numb to generate isoforms that promote neuronal cell differentiation and neurite outgrowth.
RBM4通过调节组织特异性的前体mRNA可变剪接参与细胞分化。RBM4也与小鼠胚胎大脑中的神经发生有关。利用小鼠胚胎癌细胞P19作为神经分化模型，我们观察到RBM4表达与细胞命运决定基因Numb的剪接异构体变化之间存在时间相关性。敲低RBM4影响了P19细胞中Numb外显子3和9的包含/排除。RBM4缺陷的胚胎小鼠大脑也表现出Numb前体mRNA的异常剪接。通过剪接报告基因小基因检测，我们证明RBM4促进外显子3的包含和外显子9的排除。此外，我们发现RBM4的缺失降低了神经前体基因Mash1的表达，而这种降低被RBM4诱导的包含外显子3但缺乏外显子9的Numb异构体所逆转。因此，在神经元祖细胞中诱导异位RBM4表达可增加Mash1表达并促进细胞分化。最后，我们发现RBM4对于体外皮质神经元的轴突生长也是必不可少的。RBM4缺失神经元的轴突生长缺陷被RBM4诱导的缺乏外显子9的Numb异构体所挽救。因此，我们的研究结果表明，RBM4调节Numb的外显子选择以产生促进神经元细胞分化和轴突生长的异构体。
