Our research is focused on RNA-processing proteins and RNA polymerase (RNAP)-associated transcription factors. We pioneered the structural analysis of dsRNA in complex with ribonuclease III (RNase III) enzymes. RNase III represents a family of dsRNA-specific endoribonucleases required for RNA maturation and gene regulation. Prokaryotic RNase III and eukaryotic Rnt1p, Dcr1, Drosha, and Dicer are representative members of the family. Previously, we reported a total of eleven crystal structures of a bacterial RNase III in complex with dsRNA at various catalytic stages of the enzyme, including the first structure of a catalytically meaningful RNase III-RNA complex and the structure of a catalytic stage immediately after the cleavage of the phosphodiester bond. Recently, we determined the crystal structure of a post-cleavage complex of Rnt1p from yeast, the first structure of a eukaryotic RNase III in complex with RNA in a catalytically meaningful manner. Strikingly, the structure features two rulers for substrate selection. This double-ruler mechanism represents an example of the evolution of substrate selectivity and provides a framework for understanding the catalytic mechanism of eukaryotic RNase IIIs. The worldwide effort in structural analysis of other eukaryotic RNase III enzymes resulted in several important structures, including the structures of Dicer, Dcr1, and Drosha. These structures, however, do not contain RNA and thus are not able to explain their mechanisms of action. Our structures of RNase III:dsRNA complexes greatly enhanced the significance of these important structures. Based on the protein-RNA interactions revealed by our structures of both prokaryotic and eukaryotic enzymes, models with RNA can be reliably constructed for Dicer, Dcr1, and Drosha. A model complex of Dicer with RNA explains how Dicer enzymes recognize the 2-nucleotide 3' overhang of dsRNA substrate and measure 22 nucleotides up to position the scissile bond over the cleavage site. A model complex of Dcr1 with RNA explains how homodimers of non-canonical Dicer enzymes bind cooperatively along dsRNA substrate such that the distance between active centers in adjacent homodimers is the length of 22 nt. A model complex of Drosha with RNA explains how Drosha enzymes recognize the last base pair in the basal junction of the primary microRNA substrate and measure 11 nucleotides up to position the scissile bond over the cleavage site. Our structural and mechanistic studies of biomolecular systems aim to reveal their reaction coordinates or functional cycle. To date, we have described the reaction coordinates of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK, a folate pathway enzyme essential for microorganisms but absent in mammals), the functional cycle of Era (an essential GTPase that couples cell growth with cell division), RapA (a Swi2/Snf2 protein that recycles RNA polymerase), bacterial RNase III, and yeast RNase III. Several biomolecular systems mentioned above are attractive molecular targets and structure-based drug development is an integral part of our research.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC010326-18
Application #
9556263
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
18
Fiscal Year
2017
Total Cost
Indirect Cost
Name
Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Song, He; Fang, Xianyang; Jin, Lan et al. (2017) The Functional Cycle of Rnt1p: Five Consecutive Steps of Double-Stranded RNA Processing by a Eukaryotic RNase III. Structure 25:353-363
Yu, Fei; Song, He; Wu, Yanling et al. (2017) A Potent Germline-like Human Monoclonal Antibody Targets a pH-Sensitive Epitope on H7N9 Influenza Hemagglutinin. Cell Host Microbe 22:471-483.e5
Ji, Xinhua (2016) Structural insights into cell cycle control by essential GTPase Era. Postepy Biochem 62:335-342
Kumari, Vandana; Dyba, Marzena A; Holland, Ryan J et al. (2016) Irreversible Inhibition of Glutathione S-Transferase by Phenethyl Isothiocyanate (PEITC), a Dietary Cancer Chemopreventive Phytochemical. PLoS One 11:e0163821
Kakar, Smita; Fang, Xianyang; Lubkowska, Lucyna et al. (2015) Allosteric Activation of Bacterial Swi2/Snf2 (Switch/Sucrose Non-fermentable) Protein RapA by RNA Polymerase: BIOCHEMICAL AND STRUCTURAL STUDIES. J Biol Chem 290:23656-69
Li, Shengjian; Liang, Yu-He; Mariano, Jennifer et al. (2015) Insights into Ubiquitination from the Unique Clamp-like Binding of the RING E3 AO7 to the E2 UbcH5B. J Biol Chem 290:30225-39
Blaszczyk, Jaroslaw; Lu, Zhenwei; Li, Yue et al. (2014) Crystallographic and molecular dynamics simulation analysis of Escherichia coli dihydroneopterin aldolase. Cell Biosci 4:52
Antony, Marie L; Lee, Joomin; Hahm, Eun-Ryeong et al. (2014) Growth arrest by the antitumor steroidal lactone withaferin A in human breast cancer cells is associated with down-regulation and covalent binding at cysteine 303 of ?-tubulin. J Biol Chem 289:1852-65
Maciag, Anna E; Holland, Ryan J; Kim, Youseung et al. (2014) Nitric oxide (NO) releasing poly ADP-ribose polymerase 1 (PARP-1) inhibitors targeted to glutathione S-transferase P1-overexpressing cancer cells. J Med Chem 57:2292-302
Liang, Yu-He; Lavoie, Mathieu; Comeau, Marc-Andre et al. (2014) Structure of a eukaryotic RNase III postcleavage complex reveals a double-ruler mechanism for substrate selection. Mol Cell 54:431-44

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