<em>Drosophila</em> Brain Tumor is a translational repressor
Abstract
The Drosophila brain tumor (brat) gene encodes a member of the conserved NHL family of proteins, which appear to regulate differentiation and growth in a variety of organisms. One of the founding family members, Caenorhabditis elegans LIN-41, is thought to control posttranscriptional gene expression. However, the mechanism by which LIN-41, or any other NHL protein, acts has not been clear. Using a yeast “four-hybrid” interaction assay, we show that Brain Tumor is recruited to hunchback (hb) mRNA through interactions with Nanos and Pumilio, which bind to the RNA to repress its translation. Interaction with the Nanos/Pumilio/RNA complex is mediated by the Brat NHL domain; single amino acid substitutions in this domain compromise quaternary complex assembly in vitro and hb regulation in vivo. Thus, recruitment of Brat is necessary for translational repression and the normal development of posterior embryonic pattern. In addition to regulating abdominal segmentation, previous genetic analysis has shown that Brat, Nanos, and Pumilio govern a variety of developmental processes. We examined the role of Brat in two of these processes—regulation of maternal Cyclin B mRNA in the embryo and regulation of imaginal disc development. The results of these experiments suggest that NHL domain proteins are recruited to various mRNAs by combinatorial protein–protein interactions.
Posttranscriptional regulation plays an important role in the regulation of development and metabolism (Wickens et al. 1996; Gray and Wickens 1998; Preiss and Hentze 1999). In general, this regulation is mediated by cis-acting signals in the 3′ UTR of targeted mRNAs and by proteins that recognize these signals. One well-studied case is the regulation of hunchback (hb) during early embryogenesis in Drosophila. Maternally derived hb mRNA is uniformly distributed throughout the embryo; the mRNA is translationally repressed in the posterior, giving rise to an anterior-to-posterior gradient of Hb protein (Tautz 1988). Failure of this repression results in the abnormal accumulation of Hb in the posterior, which inhibits abdominal segmentation (Hülskamp et al. 1989; Irish et al. 1989; Struhl 1989).
Two conserved RNA-binding proteins, Pumilio (Pum) and Nanos (Nos), are specifically required to repress hb translation (Barker et al. 1992; Wang et al. 1994). Pum, which is distributed uniformly throughout the embryo, is the founding member of a large family of RNA-binding proteins (Murata and Wharton 1995; Zamore et al. 1997; Zhang et al. 1997; Wharton et al. 1998). Pum binds to 32 nucleotide sites in the 3′ UTR of hb (Nos Response Elements, NREs) to regulate its translation (Murata and Wharton 1995; Zamore et al. 1997; Wharton et al. 1998). Nos, which initially is distributed as a gradient emanating from the posterior pole of the embryo, contains a conserved zinc finger that mediates nonspecific RNA binding (Curtis et al. 1997). Nos is selectively recruited into a ternary complex on hb mRNA by NRE-bound Pum (Sonoda and Wharton 1999). The mechanism by which the resulting Nos/Pum/NRE complex regulates translation is not yet understood, although deadenylation is thought to play a role (Wharton and Struhl 1991; Wreden et al. 1997).
Brain Tumor (Brat) is one of three NHL domain proteins found in Drosophila (Adams et al. 2000; Arama et al. 2000). The family name derives from three of the founding members: NCL-1, HT2A, and LIN-41 (Slack and Ruvkun 1998). All three factors have ties to RNA metabolism: the nucleoli in Caenorhabditis elegans ncl-1 mutants are enlarged (Frank and Roth 1998); HT2A was identified by virtue of interaction with the RNA-binding protein HIV Tat (Fridell et al. 1995); and posttranscriptional regulation of lin-29 mRNA is abrogated in lin-41 mutants (Slack et al. 2000). Little is known of the biological roles of other family members, and no direct molecular mechanism has been described previously for any NHL domain protein (including Brat).
In this report, we show that the NHL domain of Brat mediates its recruitment to the 3′ UTR of hb mRNA. Recruitment occurs through protein–protein interactions with RNA-bound Pum and Nos; formation of the resulting quaternary complex is essential for translational control of hb. These results suggest a general mechanism by which other NHL domain proteins may act to control posttranscriptional gene expression.
Each entry is the percentage of embryos derived from females of the indicated genotype (left) bearing the indicated number of abdominal segments (above). Forty to one-hundred embryos were scored in each case.
Acknowledgments
We thank the Fehon laboratory, H. Bogerd, B. Cullen, and C. Lehner for advice and reagents; Z. Lev for helpful discussions; C. Gardner and S. Pyle for technical assistance; S. Boyles for administrative help; and G. Johnson for media preparation. Drosophila stocks were obtained from the Bloomington and Umea Stock Centers, and information from the Berkeley Drosophila Genome Project (BDGP). R.P.W. is an Assistant Investigator of the Howard Hughes Medical Institute.
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Footnotes
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Article and publication are at www.genesdev.org/cgi/doi/10.1101/gad.870801.