Protein ligands mediate the CRM1-dependent export of HuR in response to heat shock.
AU-rich elements (AREs) located in the 3' UTRs of the messenger RNAs (mRNAs) of many mammalian early response genes promote rapid mRNA turnover. HuR, an RRM-containing RNA-binding protein, specifically interacts with AREs, stabilizing these mRNAs. HuR is primarily nucleoplasmic, but shuttles between the nucleus and the cytoplasm via a domain called HNS located between RRM2 and RRM3. We recently showed that HuR interacts with two protein ligands, pp32 and APRIL, which are also shuttling proteins, but rely on NES domains recognized by CRM1 for export. Here we show that heat shock induces increased association of HuR with pp32 and APRIL through protein-protein interactions and that these ligands partially colocalize with HuR in cytoplasmic foci. HuR associations with the hnRNP complex also increase, but through RNA links. CRM1 coimmunoprecipitates with HuR only after heat shock, and nuclear export of HuR becomes sensitive to leptomycin B, an inhibitor of CRM1. Export after heat shock requires the same domains of HuR (HNS and RRM3) that are essential for binding pp32 and APRIL. In situ hybridization and coimmunoprecipitation experiments show that LMB treatment blocks both hsp70 mRNA nuclear export and its cytoplasmic interaction with HuR after heat shock. Together, our results argue that upon heat shock, HuR switches its export pathway to that of its ligands pp32 and APRIL, which involves the nuclear export factor CRM1. HuR and its ligands may be instrumental in the nuclear export of heat-shock mRNAs.
Gallouzi
I E
IE
Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06536, USA.
Brennan
C M
CM
Steitz
J A
JA
D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D000954:Antigens, Surface; D002352:Carrier Proteins / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D051959:ELAV Proteins; D000067780:ELAV-Like Protein 1; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D006367:HeLa Cells; D018869:Heat-Shock Response; D006801:Humans; D028884:Karyopherins; D008024:Ligands; D009479:Neuropeptides / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear; D000097606:Exportin 1 Protein
D000954:Antigens, Surface; D002352:Carrier Proteins; D051959:ELAV Proteins; D000067780:ELAV-Like Protein 1; D005231:Fatty Acids, Unsaturated; D028884:Karyopherins; D008024:Ligands; D009479:Neuropeptides; D009687:Nuclear Proteins; D010750:Phosphoproteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D000097606:Exportin 1 Protein; C490341:ANP32B protein, human; C098770:ELAVL1 protein, human; C038753:leptomycin B