Nuclear export of the DEAD box An3 protein by CRM1 is coupled to An3 helicase activity.
We have recently identified the Xenopus laevis An3 protein as a bona fide substrate for the nuclear export receptor CRM1 (Exportin 1). An3 binds directly to CRM1 with high affinity via a leucine-rich nuclear export signal located in the extreme N terminus. An3 is a member of the DEAD box family of RNA helicases, which unwind RNA duplexes. RNA unwinding is coupled to hydrolysis of nucleoside triphosphates by the helicase, and the ATPase activity of several helicases is greatly stimulated by various polynucleotides. Here we report that dATP hydrolysis by An3 is stimulated approximately 6-fold by total RNA from X. laevis oocytes, whereas poly(U) RNA fails to enhance hydrolysis, suggesting the existence of a specific RNA activator for An3. Kinetic analysis reveals that a mutation within the conserved DEAD box motif reduces the rate of dATP hydrolysis by approximately 6-fold. In accordance with this, the DEAD box mutant is unable to unwind double-stranded RNA. Microinjection of the An3 DEAD box mutant into X. laevis oocytes nuclei reveals a significantly lower export rate as compared with wild-type An3 protein. This is not because the mutant has lower affinity toward CRM1, nor is it due to altered RNA binding capacity. This suggests that nuclear export of An3 protein by CRM1 is coupled to An3 helicase activity.
Askjaer
P
P
Department of Molecular and Structural Biology, University of Aarhus, DK-8000 Aarhus C, Denmark.
Rosendahl
R
R
Kjems
J
J
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism*; D053487:DEAD-box RNA Helicases; D006868:Hydrolysis; D028884:Karyopherins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D009865:Oocytes / Q000378:metabolism; D011487:Protein Conformation; D020365:RNA Helicases / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000378:metabolism*; D029867:Xenopus Proteins; D014982:Xenopus laevis; D020931:ran GTP-Binding Protein / Q000378:metabolism; D000097606:Exportin 1 Protein
D000251:Adenosine Triphosphatases; D000255:Adenosine Triphosphate; D002352:Carrier Proteins; D053487:DEAD-box RNA Helicases; D028884:Karyopherins; D009687:Nuclear Proteins; D020365:RNA Helicases; D018160:Receptors, Cytoplasmic and Nuclear; D014157:Transcription Factors; D029867:Xenopus Proteins; D020931:ran GTP-Binding Protein; D000097606:Exportin 1 Protein; C407466:DDX3X protein, Xenopus