Recruitment of Nanos to hunchback mRNA by Pumilio.
Translational regulation of hunchback (hb) mRNA is essential for posterior patterning of the Drosophila embryo. This regulation is mediated by sequences in the 3'-untranslated region of hb mRNA (the Nanos response elements or NREs), as well as two trans-acting factors-Nanos and Pumilio. Pumilio recognizes the NREs via a conserved binding motif. The mechanism of Nanos action has not been clear. In this report we use protein-protein and protein-RNA interaction assays in yeast and in vitro to show that Nanos forms a ternary complex with the RNA-binding domain of Pumilio and the NRE. Mutant forms of the NRE, Nos, and Pum that do not regulate hb mRNA normally in embryos do not assemble normally into a ternary complex. In particular, recruitment of Nos is dependent on bases in the center of the NRE, on the carboxy-terminal Cys/His domain of Nos, and on residues in the eighth repeat of the Pum RNA-binding domain. These residues differ in a closely related human protein that also binds to the NRE but cannot recruit Drosophila Nos. Taken together, these findings suggest models for how Nos and Pum collaboratively target hb mRNA. More generally, they suggest that Pum-like proteins from other species may also act by recruiting cofactors to regulate translation.
Sonoda
J
J
Howard Hughes Medical Institute (HHMI), Department of Genetics, Duke University Medical Center, Durham, North Carolina 27710, USA.
Wharton
R P
RP
D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D019521:Body Patterning / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics*; D004330:Drosophila / Q000196:embryology* / Q000235:genetics* / Q000378:metabolism; D029721:Drosophila Proteins; D018507:Gene Expression Regulation, Developmental; D006801:Humans; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D046911:Macromolecular Substances; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics*; D016601:RNA-Binding Proteins; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics*; D020798:Two-Hybrid System Techniques
D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D019476:Insect Proteins; D046911:Macromolecular Substances; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D014157:Transcription Factors; C061245:hb protein, Drosophila; C074333:pum protein, Drosophila; C070574:nos protein, Drosophila