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

• Published in: Cell reports (Cambridge) 2015-11, Vol.13 (6), p.1206-1220
• Main Authors: Loedige, Inga, Jakob, Leonhard, Treiber, Thomas, Ray, Debashish, Stotz, Mathias, Treiber, Nora, Hennig, Janosch, Cook, Kate B., Morris, Quaid, Hughes, Timothy R., Engelmann, Julia C., Krahn, Michael P., Meister, Gunter
• Format: Article
• Language: English
• Subjects: Amino Acid Motifs; Amino Acid Sequence; Animals; Binding Sites; Cell Cycle Proteins - metabolism; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Drosophila - genetics; Drosophila - metabolism; Drosophila Proteins - chemistry; Drosophila Proteins - genetics; Drosophila Proteins - metabolism; Molecular Sequence Data; Protein Binding; RNA, Messenger - metabolism
• ISSN: 2211-1247; 2211-1247
• DOI: 10.1016/j.celrep.2015.09.068

TRIM-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. [Display omitted] •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 TRIM-NHL proteins are important regulators of cell fate decisions and contact RNA targets via their NHL domains. In this study, Loedige et al. present the co-crystal structure of the NHL domain of Drosophila Brat in complex with its RNA-binding motif. This study reveals that NHL domains are widespread, sequence-specific RNA-binding modules.