Nuclear RNA-associated proteins and their relationship to the nuclear matrix and related structures in HeLa cells

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Abstract
The ultrastructure and the polypeptide composition of residual nuclear substructures including nuclear matrices, nuclear ghosts and residual envelopes were investigated by means of EM and 2-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 intranuclear matrix is apparently made of 2 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 2 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.