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 sn RNP) components U1A and U2B " is mediated by unusually long and complex nuc lear 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 nucle ocytoplasmic transport receptors, U1A/U2B " import is not inhibited by eith er 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 nonhydrolyz able adenosine triphosphate analogues not GTP can do so. U1A and U2B " impo rt in vitro does not require the addition of soluble cytosolic proteins, bu t a factor or factors required for U1A and U2B " import remains tightly ass ociated with the nuclear fraction of conventionally permeabilized cells. Th is activity can be solubilized in the presence of elevated MgCl2. These dat a suggest that U1A and U2B " import into the nucleus occurs by a hitherto u ncharacterized mechanism.