RBM4蛋白選擇性剪接能力之功能性分析

Abstract

真核細胞的基因表現往往會受到mRNA選擇性剪接的調控,進而在不同的組織中形成功能不同的蛋白質。RNA結合蛋白RBM4 具有調節RNA選擇性剪接的功能，可以影響polypyrimidine tract binding protein (PTB) 以及 α-tropomyosin等蛋白質的mRNA剪接。特別的是，動物中僅有哺乳類的RBM4蛋白質C端具有三個alanine-rich片段。過去研究指出一些人類疾病和蛋白質中alanine-rich片段的擴張有關。因此，我希望藉由以刪除RBM4的alanine-rich片段以及取其他物種的RBM4 homologs來研究alanine-rich片段的功能。然而，藉由PTB minigene作為reporter，我們發現刪除alanine-rich 片段並不影響RBM4的選擇性剪接功能。另外，far western assay的結果也顯示刪除alanine-rich 片段並不影響RBM4之self-interaction功能。 最近有研究發現一種由RBM4以及CoAA (RBM14)結合而成的蛋白- CoAZ。其N端具有CoAA的RRM而C端則同RBM4。藉由觀測CoAA以及CoAZ蛋白質在細胞中的位置以及其對於RNA選擇性剪接的影響，我們發現這兩個蛋白質和RBM4有一些類似的功能。根據in vivo splicing的結果，CoAA和CoAA分別影響了PTB 以及E1A reporter的選擇性剪接。另外，免疫螢光染色實驗顯示雖然兩者皆表現於細胞核中，但位置有些差異。CoAZ會形成特殊foci結構且四周圍繞著paraspeckle蛋白質PSF，而CoAA的表現位置則和RBM4有部分重疊。在細胞內過量表現CoAA或CoAZ則和RBM4的作用類似，會促進肌肉細胞分化的相關基因表現。這些結果顯示這三種蛋白質可能有交互作用，影響特定的RNA選擇性剪接以調控細胞分化。

In eukaryotic cells, alternative splicing of mRNA plays important roles in gene regulation and the generation of tissues-specific protein isoforms. The RNA binding motif 4 protein (RBM4) is an alternative splicing regulatory factor and involved in several RNA regulatory processes such as the splicing of polypyrimidine tract binding protein (PTB) and α-tropomyosin pre-mRNA. The C-terminal effector domain of mammalian RBM4 contains three alanine-rich stretches, whereas the homologs in other animals do not. It has been reported that expansion of the alanine tracts in a few proteins is correlated with human illnesses. Thus, we generated several mutant and homolog constructs of RBM4 and examined whether the alanine-rich domain of RBM4 affects its activity in mRNA splicing regulation and proteins interaction. Using PTB minigene as reporter, we demonstrated that deletion of alanine stretches did not impair the splicing activity of RBM4. And by using far western assay, we confirmed that the deletion of the alanine tracts did not affect the self-interaction of RBM4. Recently, a fusion protein termed CoAZ consists of the RRM-containing N-terminus of CoAA (RBM14) and the C-terminus of RBM4. By examining the localization and function of CoAA and CoAZ protein, we identified that these two proteins have a few overlapped activities as RBM4. CoAA and CoAZ exhibited potentials to change the splicing pattern of PTB or E1A reporters, and through immunofluorescence stain, we showed that both CoAA and CoAZ are nucleus proteins. CoAZ formed foci structures surrounded by paraspeckle protein PSF and CoAA partially colocalized with RBM4. Overexpression of CoAA or CoAZ promotes the muscle specific gene expression and induces myoblast differentiation as RBM4. This implies that these three proteins might have cross interaction and regulate the splicing during the differentiation process of cells.