DYNAMIC INTERACTIONS BETWEEN SPLICING SNRNPS, COILED BODIES AND NUCLEOLI REVEALED USING SNRNP PROTEIN FUSIONS TO THE GREEN FLUORESCENT PROTEIN

The U1, U2, U4/U6, and U5 small nuclear ribonucleoproteins (snRNPs) ar e subunits of splicing complexes that remove introns from mRNA precurs ors. snRNPs show a complex, transcription-dependent localization patte rn in the nucleoplasm of mammalian cells that results from their assoc iation with several distinct subnuclear structures, including interchr omatin granule clusters, perichromatin fibrils, and coiled bodies. Her e we report the analysis of snRNP localization and interaction with th e coiled body in live human cells using fusions of snRNP proteins and p80 coilin to the Green Fluorescent Protein (GFP). Despite the large s ize of the GFP tag, GFP fusions to both the core snRNP SmE and U1 spec ific U1A proteins assemble into snRNP particles and give an identical nuclear localization pattern to their endogenous counterparts. GFP-coi lin localizes specifically to coiled bodies in a transcription-depende nt fashion and provides an accurate marker for coiled bodies in a vari ety of human cell lines. Treatment of cells with the selective ser/thr -protein phosphatase inhibitor, okadaic acid, causes both GFP-snRNP an d GFP-coilin proteins to accumulate within nucleoli, but does not resu lt in nucleolar accumulation of the GFP-fused non-snRNP protein splici ng factor ASF/SF2. In all four human cell lines tested, expression of a GFP-fused p80 coilin mutant with a single serine to aspartate substi tution also caused nucleolar accumulation of splicing snRNPs and coili n, but not ASF/SF2, in structures resembling coiled bodies when viewed by electron microscopy. This work establishes an experimental system for analyzing snRNP trafficking in living cells and provides evidence that a reversible protein phosphorylation mechanism is involved in reg ulating interaction of snRNPs and coiled bodies with the nucleolus. (C ) 1998 Academic Press.