hnRNPA3通过影响mRNA出核调控hESCs多能性

hnRNPA3 regulates hESCs pluripotency through modulating mRNA export

目的探究RNA结合蛋白异质核核糖核蛋白A3(hnRNPA3)在人胚胎干细胞(hESCs)多能性维持中的生物学功能,揭示其在RNA运输过程中的新机制。方法在hESC中通过核孔复合物免疫共沉淀及细胞核质分离检测hnRNPA3与核孔互作情况及定位;抑制hnRNPA3内源表达后进行克隆形成及碱性磷酸酶染色实验,qPCR检测多能性/分化标志基因的表达变化,利用RNA荧光原位杂交技术检测对polyA^(+)RNA及OCT4 mRNA核质分布的影响;预测hnRNPA3与已知出核相关蛋白结合情况。结果hnRNPA3定位在细胞核且与核孔互作,抑制hnRNPA3的内源表达后显著抑制了hESCs的克隆形成能力(P<0.05)并导致细胞分化标志基因表达水平升高(P<0.001),同时显著抑制了polyA^(+)RNA及OCT4 mRNA的出核(P<0.001)。蛋白质互作网络预测出hnRNPA3与约1/3的已知出核相关蛋白结合。结论hnRNPA3通过影响mRNA出核调控hESCs多能性。

Objective To explore the biological function of RNA binding protein heterogeneous nuclear ribonucleoprotein A3(hnRNPA3)in the maintenance of pluripotency of human embryonic stem cells(hESCs)and to reveal its new mechanism in RNA transport.Methods The interaction of hnRNPA3 with nuclear pore and localization were examined by immunoprecipitation of nuclear pore complex and cytoplasmic separation in hESCs.After inhibiting the endogenous expression of hnRNPA3,the expression of the pluripotency/differentiation marker genes was detected by qPCR,and the effect on the nucleoplasmic distribution of polyA^(+)RNA and OCT4 mRNA was tested by RNA fluorescence in situ hybridization to predict the binding of hnRNPA3 to known RNA export associated proteins.Results hnRNPA3 was localized in the nucleus and interacted with the nuclear pore.Inhibiting the endogenous expression of hnRNPA3 significantly inhibited the cloning-formation ability of hESC(P<0.05)and resulted in the increase of the expression of cell differentiation markers(P<0.001)and significantly inhibited the export of polyA^(+)RNA as well as OCT4 mRNA(P<0.001).The protein interaction network predicted that hnRNPA3 may bind to about one-third of the known RNA export associated proteins.Conclusions hnRNPA3 regulates hESC pluripotency by impacting on mRNA export.