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) are subunits of splicing complexes that remove introns from mRNA precursors. snRNPs show a complex, transcription-dependent localization pattern in the nucleoplasm of mammalian cells that results from their association with several distinct subnuclear structures, including interchromatin granule clusters, perichromatin fibrils, and coiled bodies. Here we report the analysis of snRNP localization and interaction with the coiled body in live human cells using fusions of snRNP proteins and p80 coilin to the Green Fluorescent Protein (GFP). Despite the large size of the GFP tag, GFP fusions to both the core snRNP SmE and U1 specific U1A proteins assemble into snRNP particles and give an identical nuclear localization pattern to their endogenous counterparts. GFP-coilin localizes specifically to coiled bodies in a transcription-dependent fashion and provides an accurate marker for coiled bodies in a variety of human cell lines. Treatment of cells with the selective ser/thr-protein phosphatase inhibitor, okadaic acid, causes both GFP-snRNP and GFP-coilin proteins to accumulate within nucleoli, but does not result in nucleolar accumulation of the GFP-fused non-snRNP protein splicing factor ASF/SF2. In all four human cell lines tested, expression of a GFP-fused p80 coilin mutant with a single serine to aspartate substitution also caused nucleolar accumulation of splicing snRNPs and coilin, 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 regulating interaction of snRNPs and coiled bodies with the nucleolus.
Sleeman
J
J
Department of Biochemistry, University of Dundee, Wellcome Trust Building, Dundee, DD1 4HN, United Kingdom.
Lyon
C E
CE
Platani
M
M
Kreivi
J P
JP
Lamond
A I
AI
D002466:Cell Nucleolus / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D019319:Okadaic Acid / Q000494:pharmacology; D017354:Point Mutation; D012326:RNA Splicing; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D004791:Enzyme Inhibitors; D008164:Luminescent Proteins; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear; C068921:p80-coilin; D049452:Green Fluorescent Proteins; D019319:Okadaic Acid