title: The importin-beta binding domain of snurportin1 is responsible for the Ran- and energy-independent nuclear import of spliceosomal U snRNPs in vitro.
abstract: The nuclear localization signal (NLS) of spliceosomal U snRNPs is composed of the U snRNA's 2,2,7-trimethyl-guanosine (m3G)-cap and the Sm core domain. The m3G-cap is specifically bound by snurportin1, which contains an NH2-terminal importin-beta binding (IBB) domain and a COOH-terminal m3G-cap--binding region that bears no structural similarity to known import adaptors like importin-alpha (impalpha). Here, we show that recombinant snurportin1 and importin-beta (impbeta) are not only necessary, but also sufficient for U1 snRNP transport to the nuclei of digitonin-permeabilized HeLa cells. In contrast to impalpha-dependent import, single rounds of U1 snRNP import, mediated by the nuclear import receptor complex snurportin1-impbeta, did not require Ran and energy. The same Ran- and energy-independent import was even observed for U5 snRNP, which has a molecular weight of more than one million. Interestingly, in the presence of impbeta and a snurportin1 mutant containing an impalpha IBB domain (IBBimpalpha), nuclear U1 snRNP import was Ran dependent. Furthermore, beta-galactosidase (betaGal) containing a snurportin1 IBB domain, but not IBBimpalpha-betaGal, was imported into the nucleus in a Ran-independent manner. Our results suggest that the nature of the IBB domain modulates the strength and/or site of interaction of impbeta with nucleoporins of the nuclear pore complex, and thus whether or not Ran is required to dissociate these interactions.
authors.affiliation: Department of Cellular Biochemistry, Max Planck Institute of Biophysical Chemistry, D-37077 Göttingen, Germany.
authors.forename: Jochen
authors.initials: J
authors.lastname: Huber
authors.forename: Achim
authors.initials: A
authors.lastname: Dickmanns
authors.forename: Reinhard
authors.initials: R
authors.lastname: Lührmann
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000502:physiology*; D000255:Adenosine Triphosphate / Q000235:genetics / Q000378:metabolism; D004734:Energy Metabolism / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics; D006160:Guanosine Triphosphate / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D022022:Nuclear Pore / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000235:genetics; D039381:RNA Cap-Binding Proteins; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000235:genetics / Q000378:metabolism*; D017381:Spliceosomes / Q000235:genetics / Q000378:metabolism*; D028961:beta Karyopherins / Q000235:genetics / Q000378:metabolism*; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism; D020931:ran GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
chemical_list: D039381:RNA Cap-Binding Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear; C114090:SNUPN protein, human; D028961:beta Karyopherins; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D001616:beta-Galactosidase; D020931:ran GTP-Binding Protein