In vitro nuclear import of snRNPs: cytosolic factors mediate m3G-cap dependence of U1 and U2 snRNP transport.
We have established an in vitro snRNP nuclear import system using digitonin permeabilized somatic cells supplemented with cytosolic extracts. As model karyophiles we used digoxygenin labelled U1 snRNPs or fluorescein labelled U2 snRNPs. In vitro nuclear import of snRNPs is inhibited by anti-pore component antibodies, consistent with transport occurring through nuclear pores. This import requires ATP, cytosolic factors and a nuclear localization signal (NLS). SnRNP nuclear accumulation is saturable and distinct from protein transport. Nuclear import of snRNPs, in permeabilized NRK cells supplemented with somatic cell cytosol, requires the same NLS structures as those identified in micro-injected mammalian cells. In contrast to the situation in Xenopus oocytes, the m3G-cap is not required for in vitro nuclear import of U1 and U2 snRNPs in somatic cells. Instead, assembly of the Sm-core domain is both necessary and sufficient to mediate snRNP nuclear targeting. Interestingly, when the in vitro system was provided with cytosol from Xenopus oocytes instead of somatic cells, U1 and U2 snRNP nuclear import was provided with cytosol from Xenopus oocytes instead of somatic cells, U1 and U2 snRNP nuclear import was m3G-cap dependent. These results indicate that soluble cytosolic factors mediate the differential m3G-cap dependence of U1 and U2 snRNP nuclear import in somatic cells and oocytes. We also demonstrate the existence of a soluble cytosolic factor whose interaction with the U2 snRNP m3G-cap is both saturable and essential for U2 snRNP nuclear import in Xenopus oocytes.
Marshallsay
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Institut für Molekularbiologie und Tumorforschung, Philipps-Universität Marburg, Germany.
Lührmann
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D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D002463:Cell Membrane Permeability; D002467:Cell Nucleus / Q000378:metabolism*; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism*; D006151:Guanosine / Q000031:analogs & derivatives / Q000378:metabolism; D051379:Mice; D008845:Microinjections; D008969:Molecular Sequence Data; D009685:Nuclear Envelope / Q000378:metabolism; D009865:Oocytes; D021382:Protein Sorting Signals / Q000378:metabolism; D012315:RNA Caps; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000378:metabolism*; D017413:Ribonucleoprotein, U2 Small Nuclear / Q000378:metabolism*; D012995:Solubility; D014709:Vero Cells; D014909:Wheat Germ Agglutinins; D014981:Xenopus
D021382:Protein Sorting Signals; D012315:RNA Caps; D017412:Ribonucleoprotein, U1 Small Nuclear; D017413:Ribonucleoprotein, U2 Small Nuclear; D014909:Wheat Germ Agglutinins; D006151:Guanosine; C017778:N(2),N(2),7-trimethylguanosine