Xinhua Ji, Ph.D.

Senior Investigator

Center for Structural Biology


BG 538, RM 207 Frederick, MD 21701


Research Topics

Biomolecular Structure and Mechanism, Structure-Based Drug Design

Our research is focused on the structural biology of RNA biogenesis, with an emphasis on RNA-processing proteins and RNA polymerase-associated transcription factors, and structure-based development of therapeutic agents. The goal of structural analysis is to map the reaction trajectory or functional cycle of selected biological macromolecules, and that of drug discovery is to design, synthesize, and characterize novel anticancer and antimicrobial agents. To date, we have characterized the reaction trajectory and/or functional cycle of HPPK (a folate pathway enzyme essential for microorganisms but absent in mammals), Era (an essential GTPase that couples cell growth with cell division), RapA (a Swi2/Snf2 protein that recycles RNA polymerase), and two endonuclease III (RNase III) enzymes. Several biomolecules mentioned above are attractive molecular targets and structure-based drug development is in progress. See our Science Gallery for the scope and depth of our science. Our contribution to RNase III research is summarized below.

Members of the RNase III family, exemplified by prokaryotic RNase III and eukaryotic Rnt1p, Drosha, and Dicer, are double-stranded RNA (dsRNA)-specific endoribonucleases. They play important roles in RNA processing and maturation, post-transcriptional gene silencing, and defense against viral infection. For mechanistic studies, bacterial enzyme is a valuable model system for the entire family. We have shown how the dimerization of the RNase III endonuclease domain (RIIID) creates a catalytic valley where  two cleavage sites are located, how the catalytic valley accommodates a dsRNA in a manner such that each of the two RNA strands is aligned with one of the two cleavage sites, how the hydrolysis of each strand involves both RIIIDs, and how RNase  III uses the two cleavage sites to create the 2-nucleotide (2-nt) 3' overhangs in its products. We have also shown how magnesium is essential for the formation of a catalytically competent protein-RNA complex, how the use of two magnesium ions can drive the hydrolysis of each phosphodiester bond, and how conformational changes in both the substrate and the protein are critical elements for assembling the catalytic complex. Moreover, we have provided a stepwise mechanism for the enzyme to execute the phosphoryl transfer reaction. As informative as the bacterial enzyme for the mechanism of RNase III action, yeast Rnt1p is a valuable model system for eukaryotic RNase III enzymes. Unlike bacterial enzymes that use four catalytic side chains, eukaryotic RNase IIIs use six. It is also distinguished from bacterial enzymes that every eukaryotic RNase III has an N-terminal extension. What is more, Rnt1p exhibits a strict guanine nucleotide specificity, which is unique among RNase III enzymes. We have shown how the substrate-binding mode of Rnt1p is distinct from that of bacterial RNase III, how all six catalytic side chains are engaged in the cleavage site, how a new RNA-binding motif of Rnt1p functions as a guanine-specific clamp, and how the dsRNA-binding domain and N-terminal domain of Rnt1p function as two rulers measuring the distances between the guanine nucleotide to the two cleavage sites. This unusual mechanism of substrate selectivity represents an example of the evolution of substrate selectivity and provides a framework for understanding the mechanism of action of other eukaryotic RNase III enzymes, including Drosha and Dicer.

Over the past two decades, the worldwide effort in structural analysis of other eukaryotic RNase III enzymes resulted in several important structures, including those of Dicer and Drosha. These structures, however, either do not contain RNA at all or contain RNA but distant from the processing center; as such, they are not able to explain the mechanism of RNA processing. Nonetheless, the significance of these structures are greatly enhanced by our structures. Based on the protein-RNA interactions revealed by our structures of RNase III and Rnt1p, models with RNA can be reliably constructed for both Drosha and Dicer. A model complex of Drosha with RNA explains how Drosha enzymes recognize the last base pair in the basal junction of a primary microRNA substrate and measure 11 nucleotides up to position the scissile bond over the cleavage site. A model complex of Dicer with RNA explains how Dicer enzymes recognize the 2-nt 3' overhang of a dsRNA substrate and measure 22 nucleotides up to position the scissile bond over the cleavage site.


Dr. Ji earned his Ph.D. degree at the University of Oklahoma (1985-1990) and performed his postdoctoral research at the University of Maryland (1991-1994), where he became a Research Assistant Professor (1994-1995) before joining the National Cancer Institute (NCI), National Institutes of Health (NIH). At the NCI at Frederick, Dr. Ji established his laboratory in the ABL-Basic Research Program in 1995, moved to the Center for Cancer Research in 1999, and gained tenure in 2001 as an NIH Senior Investigator. He has been a member of the American Crystallographic Association since 1986 and of the National Honorary Chemical Society of America since 1989. Dr. Ji created the Newsletter of NIH X-ray Diffraction Special Interest Group in 2001, and he has since served as the Editor.

Selected Publications

  1. Song H, Fang X, Jin L, Shaw GX, Wang YX, Ji X. The Functional Cycle of Rnt1p: Five Consecutive Steps of Double-Stranded RNA Processing by a Eukaryotic RNase III. Structure. 2017;25(2):353-363.

  2. Liang YH, Lavoie M, Comeau MA, Abou Elela S, Ji X. Structure of a eukaryotic RNase III postcleavage complex reveals a double-ruler mechanism for substrate selection. Mol Cell. 2014;54(3):431-44.

  3. Court DL, Gan J, Liang YH, Shaw GX, Tropea JE, Costantino N, Waugh DS, Ji X. RNase III: Genetics and function; structure and mechanism. Annu Rev Genet. 2013;47:405-31.

  4. Stagno JR, Ma B, Li J, Altieri AS, Byrd RA, Ji X. Crystal structure of a plectonemic RNA supercoil. Nat Commun. 2012;3:901.

  5. Gan J, Tropea JE, Austin BP, Court DL, Waugh DS, Ji X. Structural insight into the mechanism of double-stranded RNA processing by ribonuclease III. Cell. 2006;124(2):355-66.

This page was last updated on December 8th, 2020