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 which are implicated in the mechanism of action of interferon. Their distribution was undertaken here at the ultrastructural level by the immunogold 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 scattered throughout the cell and as aggregates often associated with electron-dense doughnut-like structures showing a similar aspect whatever their subcellular localization: the cytoplasm, the nuclear envelope, and the nucleus. In general, the 2-5A synthetases were present in much more lower amounts than the PKR, probably due to the difficulty of detecting traces of proteins by electron microscopy. To circumvent this, we used a human lymphoblastoid cell line overexpressing the 69-kDa form of the 2-5A synthetase. In such cells, the synthetase was then clearly observed in both the cytoplasm and the nucleus; isolated or small clusters of gold particles were numerous in the cell mainly over the RNP fibrils of the interchromatin space, nucleolus, and ribosomes. Interestingly, gold particles were also found to be associated with the membranes of nuclear envelope and rough endoplasmic reticulum probably due to the myristilated motif of this form of 2-5A synthetase. Finally, intensely labeled electron-opaque dots sometimes associated with the nuclear pore complexes were present in the nucleus and in the cytoplasm of cells which might suggest their transport from the nucleus to the cytoplasm or reciprocally through the nuclear pore complexes. These observations indicate the wider distribution of the dsRNA-activated enzymes in the cell, thus pointing out their potential implication in as yet undetermined physiological function(s) necessary for various cellular metabolic reactions.
Besse
S
S
Laboratoire Organisation Fonctionnelle du Noyau de l'UPR 9044 CNRS, Villejuif, France.
Rebouillat
D
D
Marie
I
I
Puvion-Dutilleul
F
F
Hovanessian
A G
AG
D015088:2',5'-Oligoadenylate Synthetase / Q000032:analysis* / Q000235:genetics / Q000276:immunology / Q000502:physiology; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D002451:Cell Compartmentation; D002460:Cell Line; D002467:Cell Nucleus / Q000201:enzymology; D003593:Cytoplasm / Q000201:enzymology; D004789:Enzyme Activation; D004790:Enzyme Induction / Q000187:drug effects; D000939:Epitopes / Q000276:immunology; D006801:Humans; D007150:Immunohistochemistry; D007370:Interferon Type I / Q000494:pharmacology*; D051379:Mice; D009363:Neoplasm Proteins / Q000032:analysis / Q000502:physiology; D009685:Nuclear Envelope / Q000378:metabolism; D012330:RNA, Double-Stranded / Q000378:metabolism*; D011994:Recombinant Proteins; D013347:Subcellular Fractions / Q000201:enzymology; D013601:T-Lymphocytes; D014162:Transfection; D019892:eIF-2 Kinase / Q000032:analysis* / Q000276:immunology / Q000502:physiology
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007370:Interferon Type I; D009363:Neoplasm Proteins; D012330:RNA, Double-Stranded; D011994:Recombinant Proteins; D019892:eIF-2 Kinase; D015088:2',5'-Oligoadenylate Synthetase