Heterogeneous ribonucleoproteins (hnRNPs) have key roles in RNA biogenesis, including pre-mRNP assembly, transport and cytoplasmic localization. Here we show by biochemical fractionation of nuclear extracts and protein-protein interaction assays that the A/B-type hnRNP CBF-A is in a multiprotein complex with hnRNP A2 and A3 and hnRNP U. Using RNA affinity chromatography and gel retardation assays, CBF-A was found to bind directly to RNA trafficking sequences in the 3'-UTR of the myelin basic protein (MBP) mRNA. In primary oligodendrocytes, astrocytes, neurons, and mouse forebrain sections, CBF-A revealed a characteristic granular cytoplasmic distribution. In mouse forebrain CBF-A-positive granules were preferentially found in regions with loosely bundled myelin fibers. In cultured oligodendrocytes, CBF-A was found to be specifically associated with endogenous MBP mRNA and CBF-A gene silencing resulted in the retention of MBP granules in the cell body. Finally, immunoelectron microscopy in differentiating oligodendrocytes showed that CBF-A is located in cytoplasmic granules that are often associated with the cytoskeleton. The results suggest that CBF-A is a novel transacting factor required for cytoplasmic mRNA transport and localization.

译文

:异质核糖核蛋白(hnRNPs)在RNA生物发生中具有关键作用,包括mRNP的前装配,转运和细胞质定位。在这里,我们通过核提取物的生化分级分离和蛋白质-蛋白质相互作用分析表明,A / B型hnRNP CBF-A与hnRNP A2和A3和hnRNP U处于多蛋白复合物中。使用RNA亲和色谱和凝胶阻滞分析,CBF发现-A直接与髓鞘碱性蛋白(MBP)mRNA的3'-UTR中的RNA运输序列结合。在原发性少突胶质细胞,星形胶质细胞,神经元和小鼠前脑切片中,CBF-A显示出特征性颗粒状胞质分布。在小鼠前脑中,优先发现髓磷脂纤维束松散的区域的CBF-A阳性颗粒。在培养的少突胶质细胞中,发现CBF-A与内源MBP mRNA特异性相关,而CBF-A基因沉默导致MBP颗粒保留在细胞体内。最后,在分化少突胶质细胞中的免疫电子显微镜显示,CBF-A位于通常与细胞骨架相关的细胞质颗粒中。结果表明,CBF-A是细胞质mRNA转运和定位所需的新型交易因子。

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