Isolation and characterization of intranuclear ribonucleoprotein complexes associated with double-stranded RNA adenosine deaminase from brain cells: implications for RNA-editing and hypermutation of viral RNA in the CNS.
Double-stranded RNA adenosine deaminase (DsRAD), which converts adenosine in duplex RNA to inosine, has been implicated in editing of cellular mRNA and hypermutation of viral RNA in the central nervous system (CNS). We used subcellular fractionation to show that DsRAD in bovine brain tissues is associated with high-molecular-weight ribonucleoprotein (RNP) complexes in the nuclei. DsRAD-associated RNP complexes have apparent molecular mass of up to 500 kDa and buoyant density of 1.35 to 1.42 g cc-1 in CsCl solution. In human glioma cells, DsRAD is also found exclusively in intranuclear RNP complexes that co-sediment with the largest RNA species. These DsRAD-associated RNP complexes are dissociated by RNase A or high salt. The RNA component is not essential for DsRAD activity, and the protein component can be separated by dsRNA-affinity column, gel filtration column, and glycerol gradient into enzymatically active protein species with apparent molecular mass ranging from 120 kDa to 70 kDa in polyacrylamide gel. The bovine brain DsRAD has no apparent requirement for low-molecular-weight cofactors or metal ions. These results provide insight into the native state of DsRAD in brain cells and have interesting implications for its putative roles in RNA-editing and hypermutation of viral RNA in the CNS.
Chen
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Department of Microbiology, University of Washington School of Medicine, Seattle 98195, USA.
Hirano
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Wong
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D000243:Adenosine Deaminase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000166:cytology / Q000201:enzymology / Q000821:virology*; D002417:Cattle; D002467:Cell Nucleus / Q000737:chemistry / Q000821:virology; D005910:Glioma; D006801:Humans; D008970:Molecular Weight; D009154:Mutation / Q000502:physiology; D009474:Neurons / Q000166:cytology / Q000201:enzymology / Q000821:virology; D017393:RNA Editing / Q000502:physiology*; D012367:RNA, Viral / Q000235:genetics*; D016601:RNA-Binding Proteins; D014407:Tumor Cells, Cultured / Q000201:enzymology / Q000821:virology; D014764:Viral Proteins / Q000235:genetics
D012367:RNA, Viral; D016601:RNA-Binding Proteins; D014764:Viral Proteins; C567955:ADARB1 protein, human; D000243:Adenosine Deaminase