ULTRASTRUCTURAL-LOCALIZATION OF INTERFERON-INDUCIBLE DOUBLE-STRANDED RNA-ACTIVATED ENZYMES IN HUMAN-CELLS

The protein kinase PKR and the 2',5'-oligoadenylate (2-5A) synthetase are two interferon-induced and double-stranded RNA-activated enzymes w hich are implicated in the mechanism of action of interferon. Their di stribution was undertaken here at the ultrastructural level by the imm unogold procedure, following the use of specific monoclonal antibodies directed against PKR and 69- and 100-kDa forms of the 2-5A synthetase . These enzymes were detected as a pool of nonaggregated proteins scat tered throughout the cell and as aggregates often associated with elec tron-dense doughnut-like structures showing a similar aspect whatever their subcellular localization: the cytoplasm, the nuclear envelope, a nd the nucleus. In general, the 2-5A synthetases were present in much more lower amounts than the PKR, probably due to the difficulty of det ecting traces of proteins by electron microscopy. To circumvent this, we used a human lymphoblastoid cell line overexpressing the 69-kDa for m of the 2-5A synthetase. In such cells, the synthetase was then clear ly observed in both the cytoplasm and the nucleus; isolated or small c lusters of gold particles were numerous in the cell mainly over the RN P fibrils of the interchromatin space, nucleolus, and ribosomes. Inter estingly, gold particles were also found to be associated with the mem branes of nuclear envelope and rough endoplasmic reticulum probably du e to the myristilated moth of this form of 2-5A synthetase. Finally, i ntensely labeled electron-opaque dots sometimes associated with the nu clear pore complexes were present in the nucleus and in the cytoplasm of cells which might suggest their transport from the nucleus to the c ytoplasm or reciprocally through the nuclear pore complexes. These obs ervations indicate the wider distribution of the dsRNA-activated enzym es in the cell, thus pointing out their potential implication in as ye t undetermined physiological function(s) necessary for various cellula r metabolic reactions. (C) 1998 Academic Press.