The Crystal Structure of the NHL Domain in Complex with RNA Reveals the Molecular Basis of Drosophila Brain-Tumor-Mediated Gene Regulation

• The RNA-binding protein Brat binds several hundred mRNAs in Drosophila embryos
• Direct mRNA target binding is mediated by a highly specific RNA-binding motif
• X-ray crystallography uncovers the molecular basis of the Brat-RNA interaction
• NHL domains are RNA-binding modules with distinct RNA preferences

SummaryTRIM-NHL proteins are conserved among metazoans and control cell fate decisions in various stem cell linages. The Drosophila TRIM-NHL protein Brain tumor (Brat) directs differentiation of neuronal stem cells by suppressing self-renewal factors. Brat is an RNA-binding protein and functions as a translational repressor. However, it is unknown which RNAs Brat regulates and how RNA-binding specificity is achieved. Using RNA immunoprecipitation and RNAcompete, we identify Brat-bound mRNAs in Drosophila embryos and define consensus binding motifs for Brat as well as a number of additional TRIM-NHL proteins, indicating that TRIM-NHL proteins are conserved, sequence-specific RNA-binding proteins. We demonstrate that Brat-mediated repression and direct RNA-binding depend on the identified motif and show that binding of the localization factor Miranda to the Brat-NHL domain inhibits Brat activity. Finally, to unravel the sequence specificity of the NHL domain, we crystallize the Brat-NHL domain in complex with RNA and present a high-resolution protein-RNA structure of this fold.

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