Transportin-SR2 mediates nuclear import of phosphorylated SR proteins - PubMed (original) (raw)
Transportin-SR2 mediates nuclear import of phosphorylated SR proteins
M C Lai et al. Proc Natl Acad Sci U S A. 2001.
Abstract
Serine/arginine-rich proteins (SR proteins) are a family of nuclear factors that play important roles in both constitutive and regulated precursor mRNA splicing. The domain rich in arginine/serine (RS) repeats (RS domain) serves as both a nuclear and subnuclear localization signal. We previously identified an importin beta family protein, transportin-SR2 (TRN-SR2), that specifically interacts with phosphorylated RS domains. A TRN-SR2 mutant deficient in Ran binding colocalizes with SR proteins in nuclear speckles, suggesting a role of TRN-SR2 in nuclear targeting of SR proteins. Using in vitro import assays, we here show that nuclear import of SR protein fusions requires cytosolic factors, and that the RS domain becomes phosphorylated in the import reaction. Reconstitution of SR protein import by using recombinant transport factors clearly demonstrates that TRN-SR2 is capable of targeting phosphorylated, but not unphosphorylated, SR proteins to the nucleus. Therefore, RS domain phosphorylation is critical for TRN-SR2-mediated nuclear import. Interestingly, we found that the RNA-binding activity of SR proteins confers temperature sensitivity to their nuclear import. Finally, we show that TRN-SR2 interacts with a nucleoporin and is targeted not only to the nuclear envelope but also to nuclear speckles in vitro. Thus, TRN-SR2 may perhaps escort SR protein cargoes to nuclear subdomains.
Figures
Figure 1
Nuclear import of SR proteins in permeabilized HeLa cells. (A) Nuclear import of BSA-NLS and two GST-SR protein fusions was performed by using permeabilized HeLa cells. Fluorescein-labeled BSA-NLS or GST-fusion proteins were incubated with the HeLa cell cytoplasmic extract (cytosol) at 30°C for 30 min in the presence of an ATP-regeneration system. Reaction mixtures were then added to the permeabilized HeLa cells and the incubation continued for another 30 min at 30°C (b, e, and h) or on ice (c, f, and i). In a, d, and g, the cytosol and ATP were not added to the reaction mixtures, although the incubations with permeabilized cells were performed at 30°C. (B) Reaction mixtures containing GST-SR protein fusions were subjected to Western blot analysis by using anti-GST antibodies (Upper) and mAb 104 (Lower). Temperatures used for the two-step incubation with the cytosol as above are indicated.
Figure 2
TNR-SR2 mediates nuclear import of phosphorylated SR proteins. GST-ASF and -RS were in vitro phosphorylated or mock-treated by SRPK1. The nuclear import assay for these two GST-SR protein fusions was carried out in a bacterial extract containing TRN-SR2 (+TRN-SR2) or in mock extract (−TRN-SR2). Effects of low temperature (d and i) or of RanQ69L-GTP (0.1 mg/ml) (e and j) on import were examined.
Figure 3
RNA-binding capacity of SR proteins correlates with their temperature sensitivity to nuclear import. (A) Schematic representation of the GST fusions with ASF, FFDD, ΔRRM1, and RS domain only. Calculated molecular mass (in kDa) of unphosphorylated fusion proteins and relative UV-crosslinking efficiency of phosphorylated proteins are indicated, respectively (Right). G represents the glycine-rich hinge of ASF. (B) (a–d) Nuclear import of phosphorylated GST-ΔRRM1 and GST-FFDD was assayed at 30°C in the mock E. coli lysate or in lysate containing TRN-SR2. (e–h) Phosphorylated GST fusion SR proteins (as indicated) were subjected to nuclear import assay in the TRN-SR2-containing lysate on ice. (C) Phosphorylated GST-ASF was incubated with E. coli lysate in the absence (−) or presence (+) of RNase A under the import conditions. The reaction mixtures were sedimented on 10–30% glycerol gradient, followed by Western blot analysis by using anti-GST antibodies. GST-ASF peak fractions are indicated by arrowheads (open for mock-treated and closed for RNase-treated). Asterisks represent partially degraded GST-ASF. (D) Nuclear import of phosphorylated GST-ASF was assayed in the RNase A-treated (+RNase A) or mock-treated (−RNase A) TRN-SR2-containing extract at 30°C or on ice.
Figure 4
SR peptide interacts directly with TRN-SR2 and blocks nuclear import of GST-ASF. (A) Radiolabeled phosphorylated SR peptide (0.04 μM) was incubated with buffer alone or with 0.4 μM GST-TRN-SR2, GST-impβ, or GST-TRN. For competition experiments by using Ran, 0.8 or 2.4 μM RanGTP or RanGDP was added to appropriate reaction mixtures. One-half of each reaction mixture was analyzed by electrophoresis on a 5.5% nondenaturing gel (native gel, Upper), and the other half was fractionated by SDS/PAGE on a 20% gel (Lower) for monitoring the peptide level in the reactions, because free peptide was not detectable in the nondenaturing gel. (B) Nuclear import of GST-ASF (0.5 μM) was compared with that of BSA-NLS (0.5 μM) under control conditions (no peptide) or in the presence of phosphorylated SR peptide at a 45× molar excess over import substrate or SV40 NLS peptide at a 120× molar excess.
Figure 5
TRN-SR2 interacts with nucleoporin p62 and is targeted to nuclear speckles. (A) GST or GST-transport receptor fusions (2 μg each) were incubated with HeLa cytosol, as described in Materials and Methods. Proteins interacting with GST or GST-fusion transporters were selected by glutathione-Sepharose and detected by immunoblotting with mAb 414 (lanes 1–5) or an antibody to p62 (lanes 6–9). For competition, binding reaction mixtures contained transport buffer alone (−; lane 7), 10 μM phosphorylated SR peptide (SR; lane 8), or 5 μM RanQ69L-GTP (Ran; lane 9). Lane 1 shows 6.6% of the input into the pull-down assay. The asterisk represents a band that was detected in some but not all batches of the cytosol used. (B) Permeabilized HeLa cells were incubated with 0.6 μM GST-TRN-SR2 or GST-impβ in the presence of an ATP regeneration system. Double-label immunofluorescence was performed by using polyclonal anti-GST (a and d) and monoclonal anti-SC35 (b and e) antibodies. Merged images are shown in c and f.
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