Xinhua Ji, Ph.D.
Dr. Ji has pioneered structural analysis of double-stranded (dsRNA) in complex with ribonuclease III (RNase III) proteins, exemplified by prokaryotic RNase III and eukaryotic Rnt1p, Drosha, and Dicer. During the decade of the 2000s, his RNase III structures had provided the first structural view of dsRNA processing. Over the last decade, his Rnt1p structures had revealed a double-ruler mechanism for substrate selection. All RNase III enzymes follow a two-magnesium(II)-ion mechanism of catalysis, but eukaryotes employ more side chains in their active sites. Dr. Ji’s structures of a post-cleavage complex determined for both RNase III and Rnt1p has provided basis for accurately modeling the catalytic assemblies of Drosha and Dicer.
1) X-ray crystallography, 2) structural biology, 3) chemical biology, 4) structure-based
drug design, 5) RNA biogenesis, 6) RNase III enzymes
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.
Selected Key Publications
- Nature Communications. 10(1): 3085, 2019. [ Journal Article ]
- Nucleic Acids Res. 47(9): 4707-4720, 2019. [ Journal Article ]
Design, synthesis, and anticancer activity evaluation of irreversible allosteric inhibitors of the ubiquitin-conjugating enzyme Ube2g2.MedChemComm. 9(11): 1818-1825, 2018. [ Journal Article ]
- Wiley Interdiscip Rev RNA. 10(3): e1521, 2018. [ Journal Article ]
- Annu Rev Genet. 47: 405-431, 2013. [ Journal Article ]
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, gained tenure in 2001 as an NIH Senior Investigator, and in 2008 became a member of the Senior Biomedical Research Service (SBRS). The SBRS, established under the Public Health Service Act, was created for scientists who are considered by their peers to be outstanding in their work.
|Sudhaker Dharavath Ph.D.||Postdoctoral Fellow (Visiting)|
|Lan Jin Ph.D.||Research Fellow|
|Anjana Ram||Postbaccalaureate Fellow (CRTA)|
|Joshua S. Rose Ph.D.||Postdoctoral Fellow (CRTA)|
|Gary Shaw Ph.D.||Research Biologist|
|Genbin Shi, Ph.D.||Staff Scientist|
Dr. Joshua Rose
- Dr. Rose’s abstract/poster entitled “Development of a model protein for HIV Tat structural study and drug development” is the winner of the ACA Taylor & Francis Biomolecular Crystallography Poster Prize (2020)!
Dr. Prabakaran Ponraj
- Dr. Ponraj's article entitled “Structure of Severe Acute Respiratory Syndrome Coronavirus Receptor-binding Domain Complexed with Neutralizing Antibody” has been selected to appear in a special virtue issue: Coronaviruses (2020).
Dr. Lan Jin
- Invited talk entitled “The Molecular Mechanism of dsRNA Processing by a Bacterial Dicer” at the III International Conference on Advanced Genetics, Baltimore, Maryland (2019).
- Recipient of the Poster Award at the NCI Structural Biology Retreat for her outstanding presentation entitled “The Molecular Mechanism of dsRNA Processing by a Bacterial Dicer” (2018).
Dr. Genbin Shi
- Invited talk entitled “Structure-Based Design and Synthsis of HPPK Inhibitors” at the 17th Annual Congress of International Drug Discovery Science & Technology, Kyoto, Japan (2019).
- Invited talk entitled "Linked Purene Pterin HPPK Inhibitors Useful as Antibacterial Agents" at International Conference on Translational Medicine, San Antonio, Texas (2012).
Dr. Chao Wang
- Dr. Wang’s research article entitled “Design, synthesis, and anticancer activity evaluation of irreversible allosteric inhibitors of ubiquitin-conjugating enzyme Ube2g2” was featured on the cover of MedChemComm (2018).
Dr. Smita Kakar
- Recipient of the Poster Award at the NCI Structural Biology Retreat for outstanding presentaton of her work as shown below (2015).
- Dr. Smita Kakar was a WINNER of the NIH Fellows Award for Research Excellence (FARE) 2016 competition for her work entitled “Allosteric Activation of Bacterial Swi2/Snf2 Protein RapA by RNA Polymerase: Biochemical and Structural Studies” (2015).
Dr. Yu-He Liang
- Dr. Liang’s paper entitled “Structure of a eukaryotic RNase III post-cleavage complex reveals a double-ruler mechanism for substrate selection” was featured on the issue’s cover of Molecular Cell (2014).
- Selected to receive The Protein Society Young Investigator Award, Protein Society Young Investigator Travel Grant, and The Protein Society Finn Wold Travel Award with an invited talk entitled “Structural Basis for Sequence Specificity and Product Length of Yeast Ribonuclease III” (2012).
Dr. Jason Stagno
- WINNER of the NIH Fellows Award for Research Excellence (FARE) 2013 competition for his work entitled “Structural Basis for NusB in the Initiation of Transcription Antitermination and dsRNA Supercoiling” (2012).
- Selected to receive The American Crystallographic Association Travel Award with an invited talk entitled “Crystal structure of a plectonemic RNA supercoil” (2012).
- Recipient of The SER-CAT Young Investigator Award for his paper entitled “Structural basis for RNA recognition by NusB and NusE in the initiation of transcription antitermination” (2012).
Dr. Chao Tu
- WINNER of the NIH Fellows Award for Research Excellence (FARE) 2010 competition for his work entitled “Structure of ERA in complex with the 3′ end of 16S rRNA: Implications for ribosome biogenesis” (2009).
- Winner of The Outstanding Scientific Presentation Award for his talk entitled “ERA: a GTP-dependent Molecular Switch Recognizes the 3’ End of 16S rRNA” in the Chemistry as a Life Science Symposium at the National Cancer Institute’s Spring Research Festival at Frederick (2009).
- Selected to receive The Protein Society Young Investigator Award, Protein Society Young Investigator Travel Grant, and The Protein Society Finn Wold Travel Award with an invited talk entitled “Structure of ERA in complex with the 3′ end of 16S rRNA: Implications for ribosome biogenesis” (2009).
Dr. Jianhua Gan
- Selected as the recipient of The SER-CAT Young Investigator Award for his paper entitled “Structural insight into the mechanism of double-stranded RNA processing by ribonuclease III” (2006).
- WINNER of the NIH Fellows Award for Research Excellence (FARE) 2007 competition for his work entitled "The Mechanism of Double-Stranded RNA Cleavage by Ribonuclease III: How Dicer Dices” (2006).
Dr. Bing Xiao
- Figure 3 in Dr. Bing Xiao’s paper entitled “Crystal structure of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase, a potential target for the development of novel antimicrobial agents” has been reproduced in the college textbook BIOCHEMISTRY, the 3rd and all subsequent editions, by Donald Voet and Judith Voet (2002).
Dr. Jaroslaw Blaszczyk
- Dr. Blaszczyk’s paper entitled “Crystallographic and modeling studies of RNase III suggest a mechanism for double-stranded RNA cleavage” was featured on the issue’s cover of Structure (2001).