CHANGES IN THE SUBNUCLEAR DISTRIBUTION OF 2 RNA METABOLISM-RELATED PROTEINS CAN BE DETECTED IN NUCLEAR SCAFFOLD OR MATRIX PREPARED BY DIFFERENT TECHNIQUES

The nuclear scaffold or matrix is a mainly proteinaceous structure tho ught to act as a nucleoskeleton determining the higher order organizat ion of eukaryotic chromatin. These structures are prepared from isolat ed nuclei by a series of extraction steps involving the use of ionic d etergents or high salt, and restriction enzymes or non-specific nuclea ses to remove chromatin and other loosely bound components. Since thes e treatments are harsh and unphysiological, the question remains open as to whether or not these structures, isolated in vitro, correspond t o a nucleoskeleton existing in vivo. Recently, it has been demonstrate d that the majority of nuclear matrix proteins are involved in RNA met abolism. In this study we have employed a morphological approach invol ving the use of confocal laser scanning microscopy and indirect immuno fluorescence techniques to analyze whether two widely employed methods to prepare the nuclear scaffold or matrix can maintain the spatial di stribution of two polypeptides involved in RNA metabolism, i.e., a 105 -kDa component of spliceosomes and a ribonucleoprotein antigen. We dem onstrate that the localization of these polypeptides changes, in some cases dramatically, in the final nucleoskeletal structures when compar ed with intact cells. Only when isolated nuclei were stabilized in vit ro with the cross-linking agent sodium tetrathionate (NaTT) prior to e xtraction with 2 M NaCl and DNase I digestion, were the immunofluoresc ent patterns displayed by the nuclear matrix indistinguishable from th ose detected in intact cells. These results emphasize the usefulness o f NaTT in studying putative nucleoskeletal structures, but also show t hat the methods currently employed to prepare the nuclear scaffold or matrix may create in vitro artifacts.