SERINE THREONINE PHOSPHATASE-1 MODULATES THE SUBNUCLEAR DISTRIBUTION OF PRE-MESSENGER-RNA SPLICING FACTORS/

HeLa cell nuclei were permeabilized and reconstituted with nuclear ext ract to identify soluble nuclear factors which play a role in the orga nization of pre-mRNA splicing factors in the mammalian cell nucleus. P ermeabilized nuclei reconstituted with nuclear extract were active in transcription and DNA replication and nuclear speckles containing pre- mRNA splicing factors were maintained over several hours independent o f soluble nuclear components. The characteristic rounding up of nuclea r speckles in response to inhibition of RNA polymerase II seen in vivo was reproduced in permeabilized cells and was strictly dependent on a catalytic activity present in the nuclear extract. By inhibitor titra tion experiments and sensitivity to inhibitor 2, this activity was ide ntified as a member of the serine/threonine protein phosphatase 1 fami ly (PP1). Interference with PP1 activity affected the distribution of pre-mRNA splicing factors in transcriptionally active, permeabilized c ells, and excess PP1 activity caused increased dephosphorylation of SR proteins in nuclear speckles. These data show that the dynamic reorga nization of the mammalian cell nucleus can be studied in permeabilized cells and that PP1 is involved in the rounding up of speckles as well as the overall organization of pre-mRNA splicing factors in the mamma lian cell nucleus.