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 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 studied the atomic structures and functions of different sets of RNA-binding proteins that regulate embryonic development and maintain germline stem cells. We have also studied the atomic structure and kinetics of enzymes that participate in the maturation of transfer RNA (tRNA). 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. This work was published in Molecular Cell in November 2022. The Drosophila melanogaster protein Glorund (Glo) is critical for the proper expression and localization of nanos mRNA. Glo represses nanos (nos) translation and uses its quasi-RNA recognition motif (qRRM) domains to recognize two type of motifs within the nos mRNA: G-tract and structured UA-rich motifs. We showed previously that each of the three qRRMs is multifunctional, capable of binding to G-tract and UA-rich motifs, yet if and how the qRRMs combine to recognize the nos mRNA remained unclear. We determined solution structures of a nos RNA containing the G-tract and UA-rich motifs. The RNA structure demonstrated that a single qRRM is physically incapable of recognizing both RNA elements simultaneously. In vivo experiments further indicated that any two qRRMs are sufficient to repress nos translation. Our in vitro and in vivo data support a model whereby tandem Glo qRRMs are indeed multifunctional and interchangeable for recognition of nos G-tract or UA-rich motifs. This study, published in Nucleic Acids Research in July 2023, provides new knowledge about how multiple RNA recognition modules within an RNA-binding protein may combine to diversify the RNAs that are recognized and regulated. 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. In this fiscal year, we have continued to study the molecular mechanisms by which 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 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.