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Abstract

In Drosophila, Exuperantia (Exu) has an essential role in the determination of anterior-posterior polarity by ensuring the proper localization of bicoid (bcd) mRNA at the anterior pole of the oocyte. Exu is required during early stages of oogenesis for the establishment of bcd mRNA localization. Despite having been known for almost three decades, Exu's function in this process is still unclear. To gain insights into the molecular mechanism of bcd mRNA localization, we solved the crystal structure of Exu. The protein contains two structured domains: a 3'-5' exonuclease (exo)-like domain, and a helical sterile alpha motif (SAM)-like domain. We show that the active site in the exo-like domain is disrupted, and the protein is therefore inactive. The structure reveals that Exu forms a stable homodimer in vitro; dimerization is required in vivo, since monomeric mutants fail to rescue bcd mRNA localization in Exu-null flies. We further observed that Exu binds RNA in vitro in a non sequence-specific manner and with high affinity. Deletion of the SAM-like domain causes a dramatic drop in the RNA-binding affinity, suggesting that this domain is the major site of RNA interaction. All together, our results support the view that Exu is a RNA binding protein. Interaction with the RNA is mediated mainly by the SAM-like domain, while the exo-like domain might act as a platform to recruit additional protein factors, probably required to define target specificity in vivo. Finally, we show that Exu is a dimer; though dimerization is not required in vitro for interaction with the RNA, it is necessary in vivo for bcd mRNA localization.