The importin-beta binding domain of snurportin1 is responsible for the Ran- and energy-independent nuclear import of spliceosomal U snRNPs in vitro

J Cell Biol. 2002 Feb 4;156(3):467-79. doi: 10.1083/jcb.200108114. Epub 2002 Jan 28.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Active Transport, Cell Nucleus / physiology*
  • Adenosine Triphosphate / genetics
  • Adenosine Triphosphate / metabolism
  • Energy Metabolism / genetics
  • Genes, Reporter / genetics
  • Guanosine Triphosphate / genetics
  • Guanosine Triphosphate / metabolism
  • HeLa Cells
  • Humans
  • In Vitro Techniques
  • Nuclear Pore / metabolism*
  • Protein Structure, Tertiary / genetics
  • RNA Cap-Binding Proteins
  • Receptors, Cytoplasmic and Nuclear / genetics
  • Receptors, Cytoplasmic and Nuclear / metabolism*
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Ribonucleoprotein, U1 Small Nuclear / genetics
  • Ribonucleoprotein, U1 Small Nuclear / metabolism*
  • Spliceosomes / genetics
  • Spliceosomes / metabolism*
  • beta Karyopherins / genetics
  • beta Karyopherins / metabolism*
  • beta-Galactosidase / genetics
  • beta-Galactosidase / metabolism
  • ran GTP-Binding Protein / genetics
  • ran GTP-Binding Protein / metabolism*

Substances

  • RNA Cap-Binding Proteins
  • Receptors, Cytoplasmic and Nuclear
  • Recombinant Fusion Proteins
  • Ribonucleoprotein, U1 Small Nuclear
  • SNUPN protein, human
  • beta Karyopherins
  • Guanosine Triphosphate
  • Adenosine Triphosphate
  • beta-Galactosidase
  • ran GTP-Binding Protein