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

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

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

Jochen Huber et al. J Cell Biol. .

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.

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Figures

Figure 1.
Figure 1.
Depletion of endogenous HeLa cell transport factors by modifying the permeabilization and preincubation conditions strongly reduces the import rate of U1 snRNPs. HeLa cells were prepared for in vitro import assays by either permeabilizing with digitonin alone (A and C) or in the presence of an energy-regenerating system followed by a 15-min incubation at room temperature (B and D). Transport reactions were performed for 15 min with fluorescently labeled U1 snRNPs and import determined by fluorescence microscopy (A and B). The amount of SPN1 still bound to the cells was determined by in situ immunostaining (C and D). Bars, 20 μm.
Figure 2.
Figure 2.
Import of U1 snRNPs is dependent on exogenously added SPN1 and importin-β, but not the Sm core NLS receptor. The import of fluorescently labeled U1 snRNPs in the presence of Ran and energy was monitored in the presence of either importin-β (A), SPN1 (B), or both (C). To investigate the contribution of the Sm core receptor to the import of U1 snRNPs, unlabeled Δ5′U1 snRNPs lacking the SPN1 binding site, but still containing the Sm core NLS were added in 100-fold molar excess (D). Comparative analysis of the import of fluorescently labeled Δ5′U1 snRNPs (E). Bars, 20 μm.
Figure 3.
Figure 3.
Ran and Ran-dependent hydrolysis of NTPs are not required for SPN1-mediated translocation of U1 snRNPs through the NPC. The nuclear import of Cy3-labeled U1 snRNPs (0,04 μM) (A–E) or FLUOS-labeled BSA-NLS (0,1 μM) (F–K) in the presence of preformed adaptor–importin-β complex (0.5 μM SPN1 or 0.6 μM importin-α, respectively and 0.2 μM importin-β) was performed for 15 min at 20°C. The permeabilized cells were preincubated in T buffer (E and H) or in the presence of 2 μM Ran GDP (A–D and F–I) and 1 mM nucleotide as indicated on the left (A–C and F–H) or 20 u/ml hexokinase/glucose (D and I). Bars, 20 μm.
Figure 4.
Figure 4.
Hydrolysis of NTPs is not required for the transport of U1 snRNPs into the nucleus. HeLa cells were preincubated for 15 min in the presence of buffer (A, D, and E), 1 mM each AMP-PNP and GMP-PNP (B), or 20 U/ml hexokinase/glucose (C) before the addition of Cy3-labeled U1 snRNPs (0,04 μM) in combination with a preformed complex of 0.5 μM SPN1 and 0.2 μM importin-β (A–E) or additionally, 0.5 μM importin-β 45–462 was added (D). The cells were incubated for 15 min at room temperature (A–D) or at 0°C (E) before they were fixed. Bars, 20 μm.
Figure 5.
Figure 5.
U5 snRNPs are also imported in a Ran- and energy-independent fashion by SPN1 and importin-β. (A, B, and C) RNA and protein analysis, and integrity test of U5 snRNPs. 10 μg Cy3-labeled U5 snRNPs were centrifuged on a 1.5-ml 10–30% glycerol gradient (260,000 g, 4°C, 3 h). 150-μl fractions were taken and the RNA and protein content analyzed. (A) Proteins were fractionated on a 10/13% step, high-TEMED polyacrylamide gel, and visualized by silver staining. (B) RNA was separated on a 10% denaturing polyacrylamide gel containing 7 M urea and visualized by ethidium bromide staining. (C) The protein gel from panel A illuminated with UV light to visualize the Cy3 fluorescence label bound to the U5 snRNP proteins. Lane A, 40% of the input applied to the gradient. Lanes 1 (top) to 9 (bottom) are the fractions taken from the gradient. The molecular mass (in kD) of the U5 proteins is indicated on the left.
Figure 6.
Figure 6.
In vitro import of Cy3-labeled U5 snRNPs (0,012 μM) (A, C, and E) and FLUOS- labeled U1 snRNPs (0,04 μM) (B, D, and F). Permeabilized HeLa cells were preincubated in buffer (A, B, C, and D) or buffer containing 20 u/ml apyrase (E and F) before the simultaneous addition of Cy3-labeled U5 snRNPs and FLUOS-labeled U1 snRNPs. U snRNPs alone (A and B); U snRNPs in the presence of 0.5 μM SPN1 and 0.2 μM importin-β (preincubated for 10 min at 0°C) (C–F). Bars, 20 μm.
Figure 7.
Figure 7.
The SPN1 IBB domain is responsible for the Ran and energy independence of SPN1-mediated nuclear U snRNP import. Nuclear import of U1 snRNPs was performed with SPN1–importin-β (A–D) or IBBImpα-SPN1–importin-β (E–H) for 15 min at 20°C. During the preincubation, either buffer (A and E) or Ran-GDP + GDP (B and F), Ran-GDP + GTP (C and G), or Ran preloaded with GMP-PNP (D and H) was present. The final concentration of transport factors and nucleotides was as follows: 0,04 μM U snRNPs, 0.5 μM SPN1 or IBBImpα-SPN1, 0.2 μM importin-β, 2 μM Ran-GDP or Ran-GMPPNP, and 1 mM GDP or GTP. Bars, 20 μm.
Figure 8.
Figure 8.
The IBB domain of SPN1 alone determines the Ran- and energy-independent translocation of IBB–importin-β complexes. Import of IBBImpα–βGal (A–E) and IBBSPN1–βGal (F–K) (both at a final concentration or 0.4 μM) were analyzed after a 5-min incubation at 20°C. As indicated, 3 μM RanGDP, 20 u/ml apyrase or energy were added to the import reaction or importin-β (0,2 μM) was omitted (E and K). Bars, 20 μm.

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