Structural Studies Of Post-Transcriptional Gene Regulation

National Institute Of Environmental Health Sciences

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Abstract

RNA regulation provides a mechanism to rapidly control 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, a major focus has been on understanding how RNA regulatory proteins act in partnership to control expression of their targets. We have studied the atomic structures and functions of different sets of RNA-binding proteins that regulate germline development. We have used cryo-electron microscopy (cryo-EM) to determine high-resolution structures that show the collaboration between Dicer-1 and its partner protein Loquacious (Loqs) throughout the catalytic cycle of processing pre-microRNA to mature microRNA. We have also studied the atomic structure and kinetics of enzymes that participate in the maturation of transfer RNA (tRNA). A major focus of our group is to understand the function of Pumilio/FBF (PUF) proteins. We determined the first crystal structure of a PUF protein in complex with RNA, which allowed us to understand the RNA recognition properties of PUF proteins, and through the years we have extended these structural studies to a variety of PUF protein complexes. In this fiscal year we have continued our studies to understand how PUF proteins function in partnership with other RNA regulatory proteins. We published a manuscript in Nucleic Acids Research that demonstrates how the partner protein LST-1 (Lateral Signaling Target-1) modulates the RNA-binding activity of the PUF protein FBF-2 (fem-3 Binding Factor-2). We found that there are two sites in LST-1 that bind to FBF-2 with differing effects on overall RNA-binding affinity, and we identified the specific residues that produce the differences. A long term goal was accomplished this fiscal year to determine cryo-EM structures of Dicer enzyme complexes. We determined six cryo-EM structures of Drosophila Dicer-1 and its partner protein Loqs before binding pre-miRNA, after binding and in a catalytically competent state, after nicking one arm of the pre-miRNA, and following complete dicing and initial product release. These structures illustrate how the conformations of both proteins change through the course of a catalytic cycle to recognize characteristic features of pre-microRNA substrates. The two proteins collaborate to specify authentic pre-microRNA processing by supporting RNA strain that leads to catalysis. In addition, the Dicer-1 and Loqs protein partnership assures cleavage at the precise location to produce the correct microRNA. This specificity is essential for proper mRNA targeting. These are the first high-resolution Dicer structures to resolve the role of the partner protein in substrate recognition. We have also continued work on tRNA processing enzymes and have generated biochemical and structural data that establish the unique sequence specificity of two enzymes that generate the essential 3 tail.