The Hrp65 self-interaction is mediated by an evolutionarily conserved domain and is required for nuclear import of Hrp65 isoforms that lack a nuclear localization signal.
Hrp65, an evolutionary conserved RNA-binding protein from the midge Chironomus tentans, has a conserved DBHS (Drosophila behavior, human splicing) domain that is also present in several mammalian proteins. In a yeast two-hybrid screening we found that Hrp65 can interact with itself. Here we confirm the Hrp65 self-interaction by in vitro pull-down experiments and map the sequences responsible for the interaction to a region that we refer to as the protein-binding domain located within the DBHS domain. We also show that the protein-binding domains of Drosophila NonA and human PSF, two other proteins with conserved DBHS domains, bind to Hrp65 in the yeast two-hybrid system. These observations indicate that the protein-binding domain can mediate homodimerization of Hrp65 as well as heterodimerization between different DBHS-containing proteins. Moreover, analyses of recombinant Hrp65 by gel-filtration chromatography show that Hrp65 can not only dimerize but also oligomerize into complexes of at least three to six molecules. Furthermore, we have analyzed the functional significance of the Hrp65 self-interaction in cotransfection assays, and our results suggest that the interaction between different Hrp65 isoforms is crucial for their intracellular localization.
Kiesler
Eva
E
Department of Molecular Biology and Functional Genomics, Stockholm University, SE-10691 Stockholm, Sweden.
Miralles
Francesc
F
Ostlund Farrants
Ann-Kristin
AK
Visa
Neus
N
D021581:Active Transport, Cell Nucleus / Q000502:physiology; D000595:Amino Acid Sequence; D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism*; D019281:Dimerization; D004330:Drosophila / Q000235:genetics; D029721:Drosophila Proteins; D006801:Humans; D019476:Insect Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D000072670:PTB-Associated Splicing Factor; D011485:Protein Binding; D020033:Protein Isoforms / Q000378:metabolism; D017434:Protein Structure, Tertiary; D016601:RNA-Binding Proteins / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D020798:Two-Hybrid System Techniques
D029721:Drosophila Proteins; D019476:Insect Proteins; C070174:NONA protein, Drosophila; D009687:Nuclear Proteins; D000072670:PTB-Associated Splicing Factor; D020033:Protein Isoforms; D016601:RNA-Binding Proteins; C404122:hrp65 protein, Chironomus tentans