Abstract
Serine/arginine-rich proteins (SR proteins) are mainly involved in the splicing of precursor mRNA. RS domains are also found in proteins that have influence on other aspects of gene expression. Proteins that contain an RS domain are often located in the speckled domains of the nucleus. Here we show that the RS domain derived from a human papillomavirus E2 transcriptional activator can target a heterologous protein to the nucleus, as it does in many other SR proteins, but insufficient for localization in speckles. By using E2 as a bait in a yeast two-hybrid screen, we identified a human importin-beta family protein that is homologous to yeast Mtr10p and almost identical to human transportin-SR. This transportin-SR2 (TRN-SR2) protein can interact with several cellular SR proteins. More importantly, we demonstrated that TRN-SR2 can directly interact with phosphorylated, but not unphosphorylated, RS domains. Finally, an indirect immunofluoresence study revealed that a transiently expressed TRN-SR2 mutant lacking the N-terminal region becomes localized to the nucleus in a speckled pattern that coincides with the distribution of the SR protein SC35. Thus, our results likely reflect a role of TRN-SR2 in the cellular trafficking of phosphorylated SR proteins.
Highlights
A group of human papillomavirus E2 transcriptional activators [5, 6], RNA pol II-associated SR-like proteins [7], transcriptional coactivator PGC-1 [8], and pre-mRNA cleavage factor Im [9]
Neither of the tested domains was sufficient for speckle targeting, this result suggests that the RS domain of E2 can serve as a functional nuclear localization signals (NLSs), like the RS domains or subdomains of some splicing factors [12, 13, 17]
We show that the RS dipeptide-rich hinge of a papillomavirus E2 transactivator serves as an NLS when attached to a heterologous protein
Summary
Plasmid Construction—The -gal-hinge and -gal-RS expression vectors were constructed by insertion of a PCR product derived from the entire hinge region (amino acids 212–396) or the RS-rich subdomain (amino acids 212–346) of HPV-5 E2 into the unique KpnI site within pCH110 (Amersham Pharmacia Biotech), respectively. The resulting fusion proteins expressed in HeLa cells can be detected by monoclonal anti--gal antibody. Preparation of Anti-TRN-SR2 Antibodies—The recombinant GSTTRN-SR2C protein was overproduced in E. coli and purified by affinity chromatography via a glutathione-Sepharose column according to the manufacturer’s instruction (Amersham Pharmacia Biotech). Phosphorylated or mock-phosphorylated GST-ASF was incubated with 35 l of the HeLa cell cytoplasmic extract (equivalent to ϳ1 mg of proteins) in a 60-l mixture at 30 °C for 30 min. Bound proteins were analyzed as above, or, after blotting with anti-TRN-SR2, the filter was stained with Ponceau S. phosphorylated GST-ASF, TRN-SR2-containing extract, and RanQ69LGTP or Ran-GDP were incubated at 30 °C for 30 min. Phosphorylated or mock-phosphorylated SR peptide was added to the reaction mix containing phosphorylated GST-ASF and recombinant TRN-SR2; the pull-down assay was performed as described above
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