CYTOSTELLIN DISTRIBUTES TO NUCLEAR REGIONS ENRICHED WITH SPLICING FACTORS

Cytostellin, a apprxoximately 240 kDa phosphoprotein found in all cell s examined from human to yeast, is predominantly intranuclear in inter phase mammalian cells and undergoes continuous redistribution during t he cell cycle. Here, mammalian cytostellin is shown to localize to int ranuclear regions enriched with multiple splicing proteins, including spliceosome assembly factor, SC-35. Cytostellin and the splicing prote ins also co-localize to discrete foci (called 'dots'), which are distr ibuted throughout the cell during mitosis and part of G1. The cytostel lin that is localized to these dots resists extraction by Triton X-100 , indicating that it is tightly associated with insoluble cell structu res. All immunostainable cytostellin reappears in the nucleus before S -phase. Although cytostellin and the splicing proteins co-localize in interphase and dividing cells, cytostellin is not detected in purified spliceosomes, and it associates with six unidentified proteins, formi ng a macromolecular complex that is biochemically distinct from the pr oteins that comprise spliceosomes. This macromolecular complex is dete cted at constant levels throughout the cell cycle, and the level of cy tostellin protein remains constant during the cell cycle. Nevertheless , intranuclear cytostellin immunostaining fluctuates markedly during t he cell cycle. The monoclonal antibody (mAb) H5 epitope of cytostellin is 'masked' in serum-starved cells, but 60 minutes after serum stimul ation intense cytostellin immunoreactivity appears in the nuclear spec kles. This rapid induction of cytostellin immunoreactivity in subnucle ar regions enriched with many splicing factors, as well as accumulatio ns of RNA polymerase II (Pol II) transcripts, suggests that cytostelli n may have a function related to mRNA biogenesis.