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, we have studied the atomic structures and functions of different RNA-binding proteins that regulate embryonic development, ribosomal biogenesis, and histone protein expression. These published studies are summarized below. 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 have extended these structural studies to a variety of PUF proteins. Last year, we published a study that showed how the PUF protein Pumilio works cooperatively with the zinc-finger protein Nanos to recognize and regulate gene expression in Drosophila embryogenesis. This fiscal year we published work describing how the protein Glorund controls the expression pattern of Nanos during oogenesis. We showed that Glorund recognizes two different types of RNA elements and uses two discrete RNA-binding surfaces to recognize both RNA motifs. By engineering Glorund variants that favor a single RNA-binding mode, we showed together with Elizabeth Gaviss lab that a subset of Glorund's functions in vivo is mediated solely by the G-tract binding mode, whereas regulation of nanos mRNA requires both recognition modes. This work was published in Cell Reports. We have also used crystal structures, biochemical assays, and in vivo studies to identify new families of PUF-related proteins that operate in ribosomal biogenesis. We determined a crystal structure of yeast Nop9 protein, an essential protein in small ribosomal subunit biogenesis that represents a second sub-family of PUF-like proteins. This new PUF-like protein, like the Puf-A/Puf6 protein that we discovered previously, has different RNA recognition properties than the classical PUF proteins. Nop9 binds to a structured stem loop RNA in the pre-ribosomal RNA, and it recognizes both sequence and structural features. Together with Susan Basergas lab, we showed that Nop9 regulates correct processing of 20S pre-rRNA to 18S rRNA by inhibiting Nob1 nuclease in the nucleolus. This work was published in Nature Communications. We also recently identified how the ribosomal biogenesis factor Nop15 undergoes a structural change during the assembly of pre-ribosomes. Nop15 is an RRM protein that is essential for large ribosomal subunit biogenesis. We determined a 2.0 crystal structure of Nop15 that revealed a C-terminal -helical region obscuring its canonical RNA-binding surface. Small-angle X-ray scattering, NMR and RNA-binding analyses showed that the C-terminal residues of Nop15 are highly flexible, but essential for tight RNA binding. When we compared our crystal structure with Nop15 that is part of a cryo-electron microscopy structure of a pre-ribosome particle, we found that the C-terminal region of Nop15 undergoes a dramatic rearrangement to expose the RNA-binding surface. This rearrangement allows Nop15 to recognize the base of its stem-loop target RNA and extends a newly-formed helix to the distal loop where it forms protein interactions that support the assembly of the pre-ribosome. This work was published in Nucleic Acids Research. In collaboration with Dr. William Marzluff and Dr. Zbigniew Dominskis group, we have studied the mechanism of stem-loop binding protein (SLBP) in regulating the expression and 3 processing of histone mRNAs. Cleavage of histone pre-mRNAs at the 3 end requires SLBP and U7 snRNP, and this complex recruits other factors including FLASH and polyadenylation factors. SLBP binds to a conserved stem-loop structure upstream of the cleavage site. We showed that both human andDrosophilaSLBPs stabilize U7 snRNP on histone pre-mRNA via two regions that are not directly involved in recognizing the stem-loop structure. Moreover, we found that stabilization of U7 snRNP binding to histone pre-mRNA by SLBP requires FLASH but not the polyadenylation factors. This work was published in RNA.