In Cultured Oligodendrocytes the A/B-type hnRNP CBF-A Accompanies MBP mRNA Bound to mRNA Trafficking Sequences

Abstract

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.