Transportin-SR2 mediates nuclear import of phosphorylated SR proteins.
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.
Lai
M C
MC
Institute of Biomedical Sciences, Academia Sinica, Nankang, Taipei 11529, Taiwan.
Lin
R I
RI
Tarn
W Y
WY
D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D006367:HeLa Cells; D006801:Humans; D008562:Membrane Glycoproteins / Q000378:metabolism; D009154:Mutation; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D012313:RNA / Q000378:metabolism; D012326:RNA Splicing; D016601:RNA-Binding Proteins; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D000068103:Serine-Arginine Splicing Factors; D013696:Temperature; D028961:beta Karyopherins
D008562:Membrane Glycoproteins; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins; D010750:Phosphoproteins; D016601:RNA-Binding Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D011994:Recombinant Proteins; D028961:beta Karyopherins; C049312:nuclear pore protein p62; C119888:transportin SR; D000068103:Serine-Arginine Splicing Factors; D012313:RNA