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© The Rockefeller University Press,
0021-9525/2000//293 $5.00
The Journal of Cell Biology, Volume 148, Number 2,
, 2000 293-304
Original Article |
An Atp-Dependent, Ran-Independent Mechanism for Nuclear Import of the U1a and U2b'' Spliceosome Proteins
mattaj{at}embl-heidelberg.de
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-β 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 MgCl2. These data suggest that U1A and U2B'' import into the nucleus occurs by a hitherto uncharacterized mechanism.
Key Words: uracil-rich small nuclear ribonucleoproteins (U snRNPs) • nucleocytoplasmic transport • nuclear import • Ran GTPase • U1A protein
© 2000 The Rockefeller University Press
Abbreviations used in this paper: aa, amino acid(s); GDP, guanosine diphosphate; GST, glutathione S-transferase; hn, heterogeneous-nuclear; KNS, hnRNP K nuclear entry signal; NLS, nuclear localization signal; NPC, nuclear pore complex; Nplc, nucleoplasmin core; RBD, RNA binding domain; U sn, uracil-rich small nuclear.
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