An ATP-dependent, Ran-independent mechanism for nuclear import of the U1A and U2B" spliceosome proteins

Abstract

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-beta 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 MgCl(2). These data suggest that U1A and U2B" import into the nucleus occurs by a hitherto uncharacterized mechanism.

Figure 1

Schematic representation of the U1A and U2B′′ fusion proteins. The overall structure of U1A and U2B′′ is schematized at the top of the figure. To generate the two import substrates, U1ANLS and U2B′′NLS, the central region of U1A (aa 94–204) and U2B′′ (aa 91–146) were fused to Nplc. As a negative control (U1ANLStrunc), a truncated form of the U1ANLS (aa 94–119) was fused to Nplc. Nplc serves as a transport-deficient pentamerization module. For affinity chromatography the same fragments were fused to GST.

Figure 2

Nuclear import of fluorescein-labeled U1ANLS and U2B′′NLS in permeabilized HeLa cells. (A) Nuclear accumulation of the two proteins was compared with that of BSA-NLS in control conditions or in the presence of apyrase (1 mg/ml) or the dominant-negative importin-β mutant ΔN44 (2 μM) as indicated. (B) Effect of low temperature or of WGA (2 mg/ml) on the import of BSA-NLS (left panels) or U1ANLS (right panels).

Figure 3

Characterization of cross competition with the U1A and U2B′′ import signals. (A) Import of BSA-NLS, U1ANLS, or U2B′′NLS under either standard conditions (control) or in the presence of excess unlabeled U1ANLS or U2B′′NLS, respectively. (B) U1A import compared with classical NLS, M9, and KNS import. Fluorescently labeled U1ANLS, BSA-NLS, Nplc-M9, and Nplc-KNS were incubated with saturating amounts of unlabeled import substrates as indicated along the top of the figure.

Figure 4

U1A import does not require soluble cytosolic factors in vitro. (A) Import of U1ANLS and BSA-NLS under standard conditions (+ cytosol) or with an energy-regenerating system and buffer alone (− cytosol). (B) U1A import against a concentration gradient. U1ANLS import reaction was carried out without fixing the nuclei and images were taken at various time points as indicated. (C) Import was carried out as in B, except that either no energy was added (−ATP) or import was blocked with 2 μg/ml WGA, respectively (+WGA).

Figure 4

U1A import does not require soluble cytosolic factors in vitro. (A) Import of U1ANLS and BSA-NLS under standard conditions (+ cytosol) or with an energy-regenerating system and buffer alone (− cytosol). (B) U1A import against a concentration gradient. U1ANLS import reaction was carried out without fixing the nuclei and images were taken at various time points as indicated. (C) Import was carried out as in B, except that either no energy was added (−ATP) or import was blocked with 2 μg/ml WGA, respectively (+WGA).

Figure 5

An alternative permeabilization protocol. (A) BSA-NLS and GSTU1ANLS were incubated with nuclei that had been washed with buffers containing different MgCl₂ concentrations as indicated on the left. (B) GSTU1ANLS import into nuclei prepared by the standard permeabilization protocol (control) or after extraction in 80 mM MgCl₂ buffer. The latter nuclei were incubated in the absence or presence of extract prepared by washing nuclei in 80 mM MgCl₂ buffer (−/+ M80). (C) GST-U1ANLS import into nuclei extracted in high MgCl₂ in the absence of added extract (control) or with 80 mM MgCl₂ nuclear extract after passage over either a control column (GSTU1ANLStrunc) or a U1ANLS column (GSTU1ANLS).

Figure 6

U1A import is not inhibited by nonhydrolyzable GTP analogues or the GTP hydrolysis-deficient Ran mutant Q69L. (A) BSA-NLS and U1ANLS were incubated with permeabilized HeLa cells under standard conditions (control) or in the presence of 2 mM GTPγS. (B) Nuclei were incubated with buffer (control) or 2 μM RanQ69L before addition of labeled import substrates and Xenopus extracts.

Figure 7

U1A import requires ATP hydrolysis. Endogenous NTP levels were predepleted from permeabilized HeLa cells by incubation with apyrase. The nuclei were subsequently incubated with 2 mM UDP to buffer endogenous phosphotransfer activity. Then import substrates, either BSA-NLS (left panels) or U1ANLS (right panels) were added with either buffer alone (−NTP) or together with 500 μM of either ADP, ATP, GTP, GTPγS, or 5′adenylylimidodiphosphate as indicated.