An ATP-dependent, Ran-independent mechanism for nuclear import of the U1A and U2B" spliceosome proteins.
Nuclear import of the two uracil-rich small nuclear ribonucleoprotein (U snRNP) components U1A and U2B" is mediated by unusually long and complex nuclear localization signals (NLSs). Here we investigate nuclear import of U1A and U2B" in vitro and demonstrate that it occurs by an active, saturable process. Several lines of evidence suggest that import of the two proteins occurs by an import mechanism different to those characterized previously. No cross competition is seen with a variety of previously studied NLSs. In contrast to import mediated by members of the importin-beta family of nucleocytoplasmic transport receptors, U1A/U2B" import is not inhibited by either nonhydrolyzable guanosine triphosphate (GTP) analogues or by a mutant of the GTPase Ran that is incapable of GTP hydrolysis. Adenosine triphosphate is capable of supporting U1A and U2B" import, whereas neither nonhydrolyzable adenosine triphosphate analogues nor GTP can do so. U1A and U2B" import in vitro does not require the addition of soluble cytosolic proteins, but a factor or factors required for U1A and U2B" import remains tightly associated with the nuclear fraction of conventionally permeabilized cells. This activity can be solubilized in the presence of elevated MgCl(2). These data suggest that U1A and U2B" import into the nucleus occurs by a hitherto uncharacterized mechanism.
Hetzer
M
M
European Molecular Biology Laboratory (EMBL), D-69117 Heidelberg, Germany.
Mattaj
I W
IW
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D001324:Autoantigens; D001692:Biological Transport; D002451:Cell Compartmentation; D002463:Cell Membrane Permeability; D002467:Cell Nucleus / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives; D006367:HeLa Cells; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism*; D016601:RNA-Binding Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000378:metabolism*; D017413:Ribonucleoprotein, U2 Small Nuclear / Q000378:metabolism*; D017411:Ribonucleoproteins, Small Nuclear; D017381:Spliceosomes / Q000378:metabolism*; D020931:ran GTP-Binding Protein / Q000235:genetics / Q000378:metabolism; D055517:snRNP Core Proteins
D001324:Autoantigens; D009687:Nuclear Proteins; D016601:RNA-Binding Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear; D017413:Ribonucleoprotein, U2 Small Nuclear; D017411:Ribonucleoproteins, Small Nuclear; C526408:SNRPB2 protein, human; C075339:U1A protein; D055517:snRNP Core Proteins; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D020931:ran GTP-Binding Protein