Post-transcriptional regulation of messenger RNA (mRNA) stability and translation is an important mechanism for rapidly controlling gene expression in response to stimuli, including environmental changes. This project seeks to generate and utilize structural information to enhance our understanding of these processes with an emphasis on the importance of RNA target specificity for proper gene regulation. In this fiscal year, we have studied the role of phosphorylation to regulate the affinity of histone mRNA stem-loop binding protein (SLBP). As DNA is replicated during cell division, it must be packaged by histones. To match the level of available histones to DNA replication, histone mRNA expression is controlled by a 3-end stem-loop structure unique to replication-dependent histone mRNAs. In Drosophila, this regulation is mediated by histone mRNA stem-loop binding protein (SLBP), which has minimal tertiary structure when not bound to RNA. We have demonstrated that phosphorylation of SLBP dramatically increases binding affinity for stem-loop RNA. The phosphorylated C-terminal tail of dSLBP does not recognize RNA. Instead, increased negative charge on the C-terminal tail and stabilization of structural elements by a phosphorylation site within the RNA-binding domain promote more compact conformations that reduce the entropic barrier to binding histone mRNA. Typical PUF proteins are sequence-specific RNA-binding proteins that are important regulators of gene expression for embryonic development and germline stem cell maintenance. Beginning with determining the first crystal structure of a PUF protein in complex with RNA to recent work on the specificity of human, yeast, and C. elegans proteins, we have identified both common and unique features of RNA recognition by this family of proteins. The combination of the features in any particular protein results in a unique network of mRNAs that are regulated by that protein. We have advanced this work by identifying and studying the crystal structures and RNA target specificity of additional PUF protein family members. Crystal structures have identified two new families of PUF-related proteins and a classical PUF protein in yeast with broad RNA recognition properties.
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