Regulation of translation is a central control point in animal cells. It provides an important checkpoint in the pathways for cell growth, differentiation, or cell death. Understanding the process provides a direct link to understanding mechanisms of many diseases. However, the impact of translation regulation is most evident and best characterized in early development. We have crystallized two novel RNA binding proteins, Pumilio and Smaug that regulate translation during early embryogenesis in Drosophila. Pumilio plays an essential role in repressing the translation of hunchback mRNA, while Smaug plays a central role in regulating the translation of Nanos mRNA. Both proteins bind to specific RNA sequences in the 3'-untranslated (3'-UTR) regions of the mRNAs they regulate. Both proteins lack sequence homology to other proteins and do not contain any previously characterized RNA binding motifs. Their structures and mechanisms of RNA recognition are a complete mystery. The long-term objective of this project is to uncover the structure and mechanism of these two key translation regulators in early development. Our specific goals are 1) to determine what the Pumilio RNA binding domain looks like, 2) to determine the structure of Pumilio/Nanos/RNA ternary complex, and 3) to define the thermodynamic parameters underlying these protein-RNA and protein-protein interactions. Smaug precedes Pumilio in the cascade of translation regulatory events in early development. Our specific goals are 1) to determine what the Smaug RNA binding domain looks like, 2) to determine how it recognize the RNA hairpin, and 3) to determine the thermodynamic parameters underlying the protein-RNA interactions. We will use a combination of x-ray crystallography, in vitro and in vivo mutagenesis, and a range of other biophysical techniques to address these aims. Together, these studies promise to provide the first set of structures of mRNA translation regulators in early development.
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