Nuclear RNA-associated proteins and their relationship to the nuclear matrix and related structures in HeLa cells.
The ultrastructure and the polypeptide composition of residual nuclear substructures including nuclear matrices, nuclear ghosts, and residual envelopes were investigated by means of electron microscopy and two-dimensional gel electrophoresis. Nuclear matrices were prepared by digesting isolated nuclei with DNase I alone, followed by high-salt extraction in 2 M NaCl. Nuclear ghosts were obtained by high-salt extraction of nuclei previously digested with DNase and RNase in MgCl2-containing buffers. To prepare residual envelopes, nuclei were first digested with RNase in the presence of EDTA, then digested with Mg2+ -activated DNase I, and extracted in 2 M NaCl. The results of this comparative study support the conclusion that the intranuclear matrix is made of two distinct RNA-containing elements. One of these elements appears on ultrathin sections as a thin fibrillar network. It disappears from RNase-digested nuclei, together with numerous basic proteins, whatever the conditions of digestion. Although this element is present in extranucleolar territories, it is a major component of residual nucleoli. The second element appears as coarse-beaded fibers absent from the nucleolar areas. Its preservation in residual nuclear substructures depends on the presence of Mg2+ ions during RNase digestion of nuclei. It is enriched in two minor basic proteins of relative mass 49 000 and 70 000. The involvement of this fibrogranular element in heterogeneous nuclear RNA attachment to the nuclear matrix is discussed.
D002458:Cell Fractionation; D002467:Cell Nucleus / Q000648:ultrastructure*; D003599:Cytoskeleton / Q000648:ultrastructure; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells / Q000648:ultrastructure; D006801:Humans; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D009685:Nuclear Envelope / Q000648:ultrastructure; D012261:Ribonucleoproteins / Q000032:analysis*; D012492:Salts
D003902:Detergents; D012261:Ribonucleoproteins; D012492:Salts