Molecular mapping of the determinants involved in human Staufen-ribosome association.
The human double-stranded (ds) RNA-binding protein Staufen (hStau) is considered to have a role in RNA transport and its localization. By using sedimentation analysis on sucrose gradients, we showed that the Staufen isoform with an apparent molecular mass of 55 kDa (Stau(55)) co-fractionated with ribosomes and associated with both the 40 and 60 S ribosomal subunits, suggesting that the Staufen isoform hStau(55) plays some role in translation. To map the determinant(s) involved in this association, we generated a series of deletion mutants and analysed their subcellular distribution by cell fractionation and fluorescent immunomicroscopy. Our results demonstrated that multiple determinants promote hStau(55)-ribosome association via both an RNA-binding-dependent mechanism and protein-protein interaction. The RNA-binding activity of the ds RNA-binding protein domain 3 (dsRBD3) but not that of dsRBD4 is the first determinant. Although necessary for stable association with ribosomes, dsRBD3 alone is not sufficient and needs other determinants as co-factors. Consistently, when expressed together, dsRBD4 and the tubulin-binding domain constitute the minimal Stau(55)/ribosome protein-protein association domain. This region of Stau(55) is sufficient to associate with ribosomes independently, but requires the RNA-binding activity of dsRBD3 for complete association. Thus the results are consistent with a putative role for Stau(55) in the regulation of translation.
Department of Biochemistry, University of Montreal, P.O. Box 6128, Station Centre Ville, Montreal, Canada H3C 3J7.
Ming
M
Luo
Thomas F
TF
Duchaîne
Luc
L
DesGroseillers
D000818:Animals; D019556:COS Cells; D002458:Cell Fractionation; D003001:Cloning, Molecular; D003598:Cytoskeletal Proteins; D006801:Humans; D009154:Mutation; D011233:Precipitin Tests; D011485:Protein Binding; D020033:Protein Isoforms; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*
D003598:Cytoskeletal Proteins; D020033:Protein Isoforms; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; C492185:STAU1 protein, human; C117884:staufen protein, mammalian