In cultured oligodendrocytes the A/B-type hnRNP CBF-A accompanies MBP mRNA bound to mRNA trafficking sequences.
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.
Raju
Chandrasekhar S
CS
Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institute, Stockholm SE-171 77, Sweden.
Göritz
Christian
C
Nord
Ylva
Y
Hermanson
Ola
O
López-Iglesias
Carmen
C
Visa
Neus
N
Castelo-Branco
Goncalo
G
Percipalle
Piergiorgio
P
D020413:3' Untranslated Regions; D000818:Animals; D023081:CCAAT-Binding Factor / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D005786:Gene Expression Regulation; D006367:HeLa Cells; D034441:Heterogeneous-Nuclear Ribonucleoproteins / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D016253:Microscopy, Immunoelectron / Q000379:methods; D009836:Oligodendroglia / Q000378:metabolism*; D011485:Protein Binding; D012333:RNA, Messenger / Q000378:metabolism*
D020413:3' Untranslated Regions; D023081:CCAAT-Binding Factor; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D012333:RNA, Messenger