Stuart F.J. Le Grice, Ph.D.
Dr. Le Grice is internationally recognized for his pioneering research on retroviral and retrotransposon reverse transcriptase (RT), including development of immobilized metal chelate chromatography for rapid enzyme purification, understanding conformational dynamics by single molecule spectroscopy, and determining the crystal structure of RT-RNA/DNA complexes. These studies are combined with nucleoside analog interference approaches to understand how substrate flexibility controls RT recognition. As a member of the nucleotidyltransferase superfamily of enzymes, his work on HIV RT-associated ribonuclease H extends into developing inhibitors of structurally-related enzymes from alpha-(HSV), beta-(HCMV) and gamma-herpersviruses (KSHV).
More recently, Dr. Le Grice’s research has focused on determining the structure of viral and virus-coded lncRNAs, both in vitro and in vivo. A long-term goal is to exploit this knowledge to screen for small molecule antagonists that interact with cis-acting regulatory RNAs.
1) retrovirus replication, 2) nucleoprotein complexes, 3) antiviral strategies, 4) regulatory RNAs, 5) RNA therapeutics, 6) chemical biology
The RT Biochemistry Section focuses on nucleoprotein complexes as they relate to replication of RNA and DNA viruses of clinical significance. Projects in the laboratory have used a combination of biochemical, biophysical, structural, biological, and computational strategies to better understand processes of reverse transcription, RNA export, and genome packaging. Single-molecule spectroscopy has shown HIV-1 reverse transcriptase (RT) to be a highly dynamic enzyme, capable of sliding and changing orientation on its nucleic acid substrate, while at the same time making the novel observation that nonnucleoside RT inhibitors can influence enzyme orientation. We have generated high-resolution structures for lentiviral (HIV-1), gammaretroviral (XMRV) and LTR-retrotransposon (Ty3) RTs These structures have provided a platform for structure/function studies and drug development, with emphasis allosteric inhibitors that bind adjacent to the RNase H active site of HIV-1 RT. In an extension of this work, we are investigating whether "RNase H-like" enzymes of a- (type 1 and 2 herpes simplex virus, HSV) b- (human cytomegaolvirus, HCMV) and g-herpesviruses (KSHV) and susceptible to inhibition by chemotypes that chelate divalent metal at the active site. Long-term KSHV studies investigate the feasibility of "kick-and kill" strategies that combine latency activators with inhibitors of key viral enzymes.
Understanding RNA structure and function has taken advantage of a novel chemoenzymatic probing method (SHAPE) that can be used both in vitro and in vivo. Modifications of this technique (ai-SHAPE and SHAPE-MaP) allow us to investigate long-range tertiary interactions (i.e., kissing-loop interactions and pseudoknots) that control both genome replication and transport of unspliced RNAs. SHAPE studies are combined with computational methods designed to develop improved algorithms for predicting RNA tertiary structure. The high sensitivity of these approaches allows us to determine RNA structure in multiple cellular contexts (nuclear, cytoplasmic and virion-associated), evidence by our recent work with a long non-coding RNA (PAN) of Kaposi's sarcoma-associated herpesvirus (KSHV). This work involves collaborative interactions with several investigators in both the intramural and extramural research communities.
Finally, as our understanding of the structural basis through which cis-acting elements control virus replication improves, we have turned our focus to developing small molecule antagonists that recognize structured regulatory RNA, expanding into hepadna- (hepatitis B virus), flavi- (Dengue virus) and filoviruses (Ebola).
Research Highlights 2015-2018
Sherpa, C., Rausch, J.W., Grice, S.F.J., Hammarskjold, M.L., and Rekosh, D. (2015) The HIV-1 Rev Response Element (RRE) exists in two alternative secondary structures which promote different replication activities. Nuc. Acids Res. 43: 4676-4686.
HIV Rev forms a complex with a 351 nucleotide sequence present in unspliced and incompletely spliced human immunodeficiency virus (HIV) mRNAs, the Rev response element (RRE), to recruit the cellular nuclear export receptor Crm1 and Ran-GTP. This complex facilitates nucleo-cytoplasmic export of these mRNAs. The precise secondary structure of the HIV-1 RRE has been controversial, since studies have reported alternative structures comprising either four or five stem-loops. The published structures differ only in regions that lie outside of the primary Rev binding site. Using in-gel SHAPE, Sherpa et al. determined that the wt NL4-3 RRE comprises a mixture of both structures. To assess functional differences between these RRE ‘conformers’, conformationally locked mutants were created by site-directed mutagenesis. Subgenomic reporters, as well as HIV replication assays, demonstrated that the five stem-loop form of the RRE promotes greater functional Rev/RRE activity compared to the four stem-loop counterpart.
Zhao, H., Lin, Z., Lynn, A.Y., Varnado, B., Beutler, J.A., Murelli, R.P., Le Grice, S.F.J. and Tang, L. (2015) Two distinct modes of metal ion binding in the nuclease active site of a viral DNA-packaging terminase: insight into the two-metal-ion catalytic mechanism. Nuc. Acids Res. 43: 11003-11016.
Many dsDNA viruses encode DNA-packaging terminases, each containing a nuclease domain that resolves concatemeric DNA into genome-length units. Terminase nucleases resemble the RNase H-superfamily nucleotidyltransferases in folds, and share a two-metal-ion catalytic mechanism. Zhao et al. show that residue K428 of a bacteriophage terminase gp2 nuclease domain mediates binding of the metal cofactor Mg2+. A K428A mutation allows visualization, at high resolution, of a metal ion binding mode with a coupled-octahedral configuration at the active site, exhibiting an unusually short metal-metal distance of 2.42 Å. Such proximity of the two metal ions may play an essential role in catalysis by generating a highly positive electrostatic niche to enable formation of the negatively charged pentacovalent phosphate transition state, and provides the structural basis for distinguishing Mg2+ from Ca2+. Using a metal ion chelator β-thujaplicinol as a molecular probe, these authors observed a second mode of metal ion binding at the active site, mimicking the DNA binding state. Arrangement of the active site residues differs drastically from those in RNase H-like nucleases, suggesting a drifting of the active site configuration during evolution. The two distinct metal ion binding modes unveiled mechanistic details of the two-metal-ion catalysis at atomic resolution.
Crawford, D.W., Blakeley,B.D., Chen,P.-C. Sherpa,C., Le Grice, S.F.J., Laird-Offringa, I.A. and McNaughton, B.R. (2016) An Evolved RNA Recognition Motif That Suppresses HIV-1 Tat/TAR-Dependent Transcription. ACS Chemical Biology 11: 2206-2215.
Potent and selective recognition and modulation of disease-relevant RNAs remain a daunting challenge. Crawford et al. used yeast display and saturation mutagenesis of established RNA-binding regions in U1A to identify new synthetic proteins that potently and selectively bind TAR RNA. The best candidate has truly altered, not simply broadened, RNA-binding selectivity, binding TAR with subnanomolar affinity (apparent dissociation constant of ∼0.5 nM), but does not appreciably bind the original U1A RNA target (U1hpII). The evolved protein specifically recognizes the TAR RNA hairpin in the context of the HIV-1 5′-untranslated region, inhibits the interaction between TAR RNA and an HIV trans-activator of transcription (Tat)-derived peptide, and suppresses Tat/TAR-dependent transcription. Proteins described in this work are among the tightest TAR binding reagents reported to date and thus have potential utility as therapeutics and basic research tools. These findings also demonstrate how a naturally occurring RNA recognition motif can be dramatically resurfaced through mutation, leading to potent and selective recognition and modulation of disease-relevant RNA.
Abulwerdi, F.A., Shortridge, M.D., Sztuba-Solinska, J., Wilson, R., Le Grice, S.F.J., Varani, G and Schneekloth, J.S. (2016) Development of Small Molecules with a Highly Selective and Non-Canonical Binding Mode to HIV-1 TAR RNA. J. Med. Chem. 59: 11148-11160
Small molecules that bind to RNA potently and specifically are relatively rare. The study of molecules that bind to the HIV-1 transactivation response (TAR) hairpin, a cis-acting HIV genomic element, has provided an important model system for RNA targeting chemistry. Abulwerdi et al. report the synthesis, biochemical, and structural evaluation of a series of molecules that bind to HIV-1 TAR RNA. A promising analogue (15) retained the TAR binding affinity of the initial hit and displaced a Tat-derived peptide with an IC50 of 40 μM. NMR characterization of a soluble analogue (2) revealed a noncanonical binding mode for this class of compounds. Finally, evaluation of 2 and 15 by SHAPE indicated specificity in binding to TAR within the context of an in vitro-synthesized 365-nt HIV-1 5′-untranslated region (UTR). These compounds exhibit a novel and specific mode of interaction with TAR, providing important suggestions for RNA ligand design.
Finding the target site and associating in a specific orientation are essential tasks for DNA-binding proteins. In order to make the target search process as efficient as possible, proteins should not only rapidly diffuse to the target site but also dynamically explore multiple local configurations before diffusing away. Protein flipping is an example of this second process that has been observed previously, but the underlying mechanism of flipping remains unclear. Ganji et al. probed the mechanism of protein flipping at the single molecule level, using HIV-1 reverse transcriptase (RT) as a model system. To test the effects of long-range attractive forces on flipping efficiency, salt concentration and macromolecular crowding conditions were varied. As expected, increased salt concentrations weaken the binding of RT to DNA while increased crowding strengthens the binding. Moreover, when flipping kinetics were analyzed (i.e. the rate and probability of flipping at each condition) this phenomenon was more efficient when RT bound more strongly. Such data is consistent with a view that DNA bound proteins undergo multiple rapid re-binding events (or short hops) that allow the protein to explore other configurations without completely dissociating.
Sztuba-Solinska, J., Diaz, L., Kumar, M.R, Kolb, G., Wiley, M.R., Jozwik, L., Kuhn, J.H., Palacios, G., Radoshitzky, S.R., Le Grice, S.F.J. and Johnson, R.F. (2016). A small stem-loop structure of the Ebola virus trailer is essential for replication and interacts with heat-shock protein A8. Nuc. Acids Res. 44: 9831-9846.
Ebola virus (EBOV) is a single-stranded negative-sense RNA virus belonging to the Filoviridae family. The leader and trailer non-coding regions of the EBOV genome likely regulate its transcription, replication, and progeny genome packaging. cis-acting RNA signals involved in RNA–RNA and RNA–protein interactions that regulate replication of eGFP-encoding EBOV minigenomic RNA were investigated and identified heat shock cognate protein family A (HSC70) member 8 (HSPA8) as an EBOV trailer-interacting host protein. Mutational analysis of the trailer HSPA8 binding motif revealed that this interaction is essential for EBOV minigenome replication. SHAPE analysis of the secondary structure of the EBOV minigenomic RNA indicates formation of a small stem-loop composed of the HSPA8 motif, a 3΄ stem-loop that is similar to a previously identified structure in the replicative intermediate (RI) RNA and a panhandle domain involving a trailer-to-leader interaction. Results of minigenome assays and an EBOV reverse genetic system rescue support a role for both the panhandle domain and HSPA8 motif 1 in virus replication.
Sztuba-Solinska, J., Rausch, J.W., Smith, R., Miller, J.T., Whitby, D and Le Grice, S.F.J. (2017). Kaposi’s sarcoma-associated herpesvirus polyadenylated nuclear RNA: a structural fold for nuclear, cytoplasmic and viral proteins. Nuc. Acids Res. 45: 6805-6821.
Kaposi's sarcoma-associated herpes virus (KSHV) polyadenylated nuclear (PAN) RNA facilitates lytic infection, modulating the cellular immune response by interacting with viral and cellular proteins and DNA. Although numerous nucleoprotein interactions involving PAN have been implicated, our understanding of binding partners and PAN RNA binding motifs remains incomplete. Sztuba-Solinska et al. used SHAPE-mutational profiling (SHAPE-MaP) to probe PAN in its nuclear, cytoplasmic or viral environments or following cell/virion lysis and removal of proteins. This study characterized and put into context discrete RNA structural elements, including the cis-acting Mta responsive element and expression and nuclear retention element (1,2). By comparing mutational profiles in different biological contexts, sites on PAN either protected from chemical modification by protein binding or characterized by a loss of structure were identified. While some protein binding sites were selectively localized, others were occupied in all three biological contexts. Individual binding sites of select KSHV gene products on PAN RNA were also identified in in vitro experiments. This work provides a broad framework for understanding the roles of PAN RNA in KSHV infection.
Sherpa C, Rausch JW and Le Grice SFJ: Structural characterization of maternally expressed gene 3 RNA reveals conserved motifs and potential sites of interaction with polycomb repressive complex 2. Nuc. Acids Res., in press, 2018.
Long non-coding RNAs (lncRNAs) have emerged as key players in gene regulation. However, our incomplete understanding of the structure of lncRNAs has hindered molecular characterization of their function. Maternally expressed gene 3 (Meg3) lncRNA is a tumor suppressor that is downregulated in various types of cancer. Mechanistic studies have reported a role for Meg3 in epigenetic regulation by interacting with chromatin-modifying complexes such as the polycomb repressive complex 2 (PRC2), guiding them to genomic sites via DNA-RNA triplex formation. Resolving the structure of Meg3 RNA and characterizing its interactions with cellular binding partners will deepen our understanding of tumorigenesis and provide a framework for RNA-based anti-cancer therapies. In this publication, Sherpa et al. characterized the architectural landscape of Meg3 RNA and its interactions with PRC2 from a functional standpoint.
Okamoto K, Rausch JW, Wakashin H, Yulong F, Chung J-Y, Dummer PD, Shin M, Chandra P, Suzuki K, Shrivastav S, Rosenberg AZ, Hewitt, SM, Ray P, Noiri E, Le Grice SFJ, Hoek M, Han Z, Winkler CA, Kopp JB. APOL1 risk allele RNA contributes to renal toxicity injury by activating protein kinase R. Communications Biology, 2018, in press.
APOL1 risk alleles associate with chronic kidney disease in African Americans, but the mechanisms remain to be fully understood. We show that APOL1 risk alleles activate protein kinase R (PKR) in cultured cells and transgenic mice. This effect is preserved when a premature stop codon is introduced to APOL1 risk alleles, suggesting that APOL1 RNA but not protein is required for the effect. Podocyte expression of APOL1 risk allele RNA, but not protein, in transgenic mice induces glomerular injury and proteinuria. Structural analysis of the APOL1 RNA shows that the risk variants possess secondary structure serving as a scaffold for tandem PKR binding and activation. These findings provide a mechanism by which APOL1 variants damage podocytes and suggest novel therapeutic strategies.
Miller, J.T., Zhao, H., Masaoka, T., Varnado, T., Cornejo Castro, E., Marshall, V.A., Kouhestani, K., Lynn, A.Y., Aron, K.E., Xia, A., Beutler, J.A., Hirsch, D.R., Tang, L., Whitby, D., Murelli, R.P. and Le Grice, S.F.J. Sensitivity of the C-terminal nuclease domain of Kaposi’s sarcoma herpesvirus to two classes of active site ligands. Antimicrobial Agents and Chemotherapy 62: 2018, in press.
Kaposi's sarcoma-associated herpesvirus (KSHV), the etiological agent of Kaposi's sarcoma, belongs to the Herpesviridae family, whose members employ a multicomponent terminase to resolve nonparametric viral DNA into genome-length units prior to their packaging. Homology modeling of the ORF29 C-terminal nuclease domain (pORF29C) and bacteriophage Sf6 gp2 have suggested an active site clustered with four acidic residues, D476, E550, D661, and D662, that collectively sequester the catalytic divalent metal (Mn2+) and also provided important insight into a potential inhibitor binding mode. Using this model, wild-type pORF29C and variants with substitutions at the proposed active-site residues were expressed, purified and characterized. Differential scanning calorimetry demonstrated divalent metal-induced stabilization of wild-type (WT) and D661A pORF29C, consistent with which these two enzymes exhibited Mn2+-dependent nuclease activity, although the latter mutant was significantly impaired. Thermal stability of WT and D661A pORF29C was also enhanced by binding of an α-hydroxytropolone (α-HT) inhibitor shown to replace divalent metal at the active site. For the remaining mutants, thermal stability was unaffected by divalent metal or α-HT binding, supporting their role in catalysis. pORF29C nuclease activity was also inhibited by two classes of small molecules reported to inhibit HIV RNase H and integrase, both of which belong to the superfamily of nucleotidyltransferases. Finally, α-HT inhibition of KSHV replication suggests ORF29 nuclease function as an antiviral target that could be combined with latency-activating compounds as a shock-and-kill antiviral strategy.
Selected Key Publications
- Communications Biology. 1: 188, 2018. [ Journal Article ]
Structural characterization of Maternally Expressed Gene 3 RNA reveals conserved motifs and potential sites of interaction with Polycomb Repressive Complex.Nucleic Acids Res. 46(19): 10432-10447, 2018. [ Journal Article ]
- RNA Biol.. 15: 13-16, 2018. [ Journal Article ]
Combining asymmetric 13C-labeling and isotopic filter/edit NOESY: a novel strategy for rapid and logical RNA resonance assignment. .Nuc. Acids Res.. 45: e146, 2017. [ Journal Article ]
- Nat. Struct. Mol. Biol. 20: 230-6, 2013. [ Journal Article ]
Stuart Le Grice received his Ph.D. from the Department of Biochemistry, University of Manchester, UK, in 1976, where he studied RNA polymerase of Escherichia coli. After postdoctoral training in the United Kingdom, Germany, and the United States, he was appointed Senior Scientist at Hoffmann La Roche, Basel, Switzerland, where he worked from 1984 to 1990 evaluating HIV-1 and HIV-2 enzymes as therapeutic targets. In 1990, he joined the faculty in the Division of Infectious Diseases, Department of Medicine, Case Western Reserve University (CWRU), Cleveland, OH. Recruited as an Associate Professor of Medicine, he was awarded tenure in 1992, and in 1995 was promoted to Professor of Medicine, Biochemistry, and Oncology. From 1994 to 1999, he served as Director of the NIH-funded CWRU Center for AIDS Research, during which time he was designated a CWRU “Million Dollar Professor” in recognition of his NIH funding. Dr. Le Grice joined the National Cancer Institute in 1999 and in 2005 was appointed to the Senior Biomedical Research Service. In 2006, he was appointed Head of the CCR Center of Excellence in HIV/AIDS & Cancer Virology. In addition to serving on the Editorial Board of the Journal of Biological Chemistry, Dr. Le Grice has been an ad hoc (1990-1999) and permanent Study Section member of NIH AIDS review panels (2000-2004), as well as an ad hoc reviewer for multiple international funding agencies.
Dr. Le Grice was designated a CCR “Mentor of Merit” in 2007 and 2009, and has been recipient of the NIH Award of Merit (2009) and two NIH Director’s Awards (2012, 2015). In 2015, Dr. Le Grice received the DHHS Career Achievement Award, recognizing his “outstanding administrative and scientific contributions to furthering the national and international mission of the National Institutes of Health.”
|Ellen Bak||Postbaccalaureate Fellow (IRTA/CRTA)|
|Samantha Gacasan Ph.D.||Postdoctoral Fellow (CRTA)|
|Stefano Ginocchio||Postbaccalaureate Fellow|
|Regan Leblanc Ph.D.||Postdoctoral Fellow (CRTA)|
|Jennifer T. Miller Ph.D.||Technical Laboratory Manager|
|Julie M. Nyman||Postbaccalaureate Fellow (CRTA)|
|Jason W. Rausch, Ph.D.||Staff Scientist|
|Chringma Sherpa Ph.D.||Postdoctoral Fellow (Visiting)|
|Monalisa Swain Ph.D.||Scientist I (Leidos)|
Le Grice Lab Awards and Recognition
- Federal Technology Transfer Award
Stuart Le Grice received a Federal Technology Transfer Award based on submission of US Patent Applications 62/598807 entitled "Chemical entities for lytic activation of KSHV and therapeutic targeting of viral enzymes/proteins", 62/685026 entitled “Cancer Treatment Methods”, and 62/685145 entitled “Methods of Treating HBV”
- RNA Society-Sponsored RNA Salon
Stuart Le Grice and Nathan Baird (University of the Sciences, Philadelphia, PA) successfully re-competed for an RNA Society RNA Salon Award to establish the “Noncoding RNA and Therapeutic Discovery Interest Group”
- Sallie Rosen Kaplan (SRK) Fellowship
Chringma Sherpa received an SRK Fellowship, a highly competitive, unpaid, annual, one-year program that provides additional mentoring opportunities, networking, seminars, and workshops to help prepare NCI’s female postdoctoral fellows for the competitive nature of the job market and help them to transition to independent research careers. The program addresses the fact that, based on recent observational, longitudinal, and intervention studies, women in science are significantly more likely to leave the science field earlier than men, specifically at the transition from a mentored scientist to an independent investigator.
- Directors Intramural Innovation Award
Fardokht Abulwerdi received a Director’s Intramural Innovation Award of $10,000 based on her proposal to identify small molecule antagonists of the MALAT1 ENE triple helix.
- Fellows Award in Research Excellence (FARE)
Chringma Sherpa was a FARE awardee based on her work deciphering the in vivo structure of the lncRNA, Meg3.
- RNA Society-Sponsored RNA Salon
Stuart Le Grice and Nathan Baird (University of the Sciences, Philadelphia, PA) were co-recipients of an RNA Society RNA Salon Award to establish the “Noncoding RNA and Therapeutic Discovery Interest Group”
- Assistant Professorship – Auburn University
Joanna Sztuba-Solinska joined the Department of Biological Sciences, Auburn University, as an Assistant Professor.
- FLEX Award
Stuart Le Grice, Jay Schneekloth and Tom Misteli were recipients of a 1-year FLEX award extension to investigate small molecule targeting of regulatory viral and cellular RNAs
- DHHS Secretary’s Career Achievement Award
Stuart Le Grice received the DHHS Secretary’s Career Achievement Award, in recognition of “Outstanding administrative and scientific contributions to furthering the national and international mission of the National Institutes of Health”.
- FLEX Award
Stuart Le Grice, Jay Schneekloth and Tom Misteli were recipients of a 3-year FLEX award to investigate small molecule targeting of regulatory viral and cellular RNAs
- NICBR Collaborative Project Award
Joanna Sztuba-Solinska received a $10,000 award to work with USAMRIID under the National Interagency Confederation for Biological Research (NICBR). The title of her research proposal is "Therapeutic targeting of structural motifs of the Dengue virus RNA genome."
- International Fellow of NCI at Frederick
Joanna Sztuba-Solinska was honored as one of five “International Fellows of NCI at Frederick”. She received this award in recognition of her achievements and contributions toward the mission of the NCI at Frederick community. As a postdoctoral fellow in the HIV DRP, she has focused her research on the exploration of RNA structural elements in the development of small-molecule therapeutic intervention of cancers and viral diseases. Details of her research interests and achievements are currently on display in the NCI at Frederick Conference Center.
- Fellows Award in Research Excellence (FARE)
Takashi Masaoka was a FARE awardee based on his work to develop inhibitors of retroviral and herpesvirus enzymes
- SAIC Corporate Award
Marion Bona was one of four SAIC-Frederick employees who were recognized among the winners of the annual SAIC Corporate Science Technology Fellows Council awards for publications that are considered to represent the very best of SAIC technical publications in the past year. She and Eckart Bindewald (CCR Nanobiology Program) won in the Physical Sciences category for their article, “Correlating SHAPE Signatures with Three-dimensional RNA Structures,” published in RNA (17:1688–1696, Cold Spring Laboratory Press, 2011). In selecting Bindewald and Bona’s article, the council commented that their paper was a “well-written paper documenting a new approach to interpreting SHAPE data, which will lead to a better understanding of RNA structure.”
- NCI Director's Intramural Innovation Award
Sabrina Lusvarghi received a $10,000 NCI Director's Intramural Innovation Award for her application entitled "Incorporation of Cross-linkable Unnatural Amino Acids for Identifying Binding Partners for XMRV Proteins." Site-specific incorporation of unnatural amino acids into proteins is a growing area of chemical biology and biochemistry, and the availability of unnatural amino acids with novel biophysical properties is constantly widening the scope of these strategies. The goal of Dr. Lusvarghi’s proposal involves incorporating photo-crosslinkable unnatural amino acids into viable viruses, an approach that is currently unprecedented. The novelty of this strategy is that the photo-crosslinkable viruses can be used to study protein-protein interactions in their most-biologically relevant context. Moreover, previously unknown binding partners identified using this approach could serve as targets for novel antiviral therapies. Finally, success in this initial investigation will likely spawn parallel studies to identify and characterize binding partners of other XMRV proteins or proteins in related retroviruses of public health significance.
- John T. Carey Memorial Lecture
Stuart Le Grice was nominated to give the Annual John. T. Carey Memorial Lecture at Case Western Reserve University, Cleveland Ohio.
- Directors Intramural Innovation award
Michal Legiewicz received a $10,000 NCI Director's Intramural Innovation Award for his research proposal entitled "Structural Responses in Tumor Suppressor Messenger RNAs Induced by micro-RNA miR-21 in Breast Cancer." Dr. Legiewicz initiated this project to investigate how the structure of messenger RNA responds to downregulation by microRNA. While down-regulation by miR-21 and its binding to target mRNAs have been tested in functional and binding assays, the structural response of target mRNAs is unknown. Dr. Legiewicz's study employs a novel high-throughput RNA-probing technology to monitor structural rearrangements within 3'- and 5'-UTRs in response to miRNA modulation. He is investigating whether the binding of miR-21 invokes translational inhibition by rearranging a local subdomain or by altering the architecture of long-range interactions within its target mRNAs (which range in size from ~2000-4000 nucleotides). In order to explore the hypothesis that miR-21 triggers a common structural response in target mRNA causing translational inhibition, Dr. Legiewicz selected three mRNAs for his study. Each is a direct target of miR-21 and is downregulated in breast cancer. The results of his study will reveal a novel mechanistic basis for miRNA-directed regulation of gene expression and new molecular targets critical in designing more potent therapies against breast cancer and against cancer in general. This award is the second that Dr. Legiewicz has received through the NCI Director's Intramural Innovation Award Program (see the text below for a description of his first award).
- NCI-Frederick Student Science Jeopardy Tournament
Brittany Ashe and David Kaiser-Jones won the 3rd Annual NCI-Frederick Scientific Library Student Science Jeopardy Tournament in 2009, competing against 11 other 2-student teams in the traditional Jeopardy "answer and question" format. Brittany and David are students working in the Le Grice lab with mentors Jason Rausch and Michal Legiewicz, respectively.
- NCI Mentor of Merit Award
Stuart Le Grice was nominated for the NCI Outstanding Mentor Award and was one of the highest rankings in a competitive review in which nominees were "judged on their records as mentors; their accessibility to trainees; their ability to communicate and provide instruction and constructive feedback; their capacity to provide an environment conducive to science and learning; their propensity to give credit to trainees and promote visibility of their work; and their attention to the career development needs of those they mentor."
- NIH Merit Award
Stuart Le Grice was nominated and selected to receive a 2009 NIH Merit Award with Robert Yarchoan (HIV and AIDS Malignancy Branch, NCI). Robert Wiltrout, Director of the Center for Cancer Research, NCI, nominated Drs. Le Grice and Yarchoan for this Group Award, titled HIV/AIDS and HIV Malignancy Leadership Group, for leadership in promoting and supporting research in HIV/AIDS and HIV-associated malignancies in the NCI.
- Fellows Award in Research Excellence (FARE)
Michaela Wendeler was a FARE awardee based on her work using unnatural amino acids to study protein structure and function.
Yi Wang received a FARE award for her studies on XMRV RT.
- Assistant Professorship – Meharry Medical College
Chandravanu Dash joined the faculty Meharry Medical School, Nashville, Tennessee as an Assistant Professor.
- Directors Intramural Innovation award
Michal Legiewicz was co-awardee of $10,000 from the NCI Director's Intramural Innovation Award Program in 2008 for the proposal "Structural Determinants Within the 5'-UTR of Cancer-Relevant mRNAs Regulated at the Level of Translation." The NCI Director's Intramural Innovation Award Program is designed to support the development of highly innovative approaches and technology aimed at significant cancer-related problems. Deregulation of protein synthesis is an early event in cancer progression. In recent years, considerable effort has been focused on translation as a molecular target for both cancer prevention and therapy. The novel tumor suppressor PDCD4 functions by inhibiting the RNA helicase activity of the eukaryotic initiation factor eIF4A and hence the efficient translation of specific oncoproteins. Applying the innovative RNA mapping technology SHAPE to determine structural signatures within the 5'-UTR of various mRNAs will explain why only select mRNAs are targets of PDCD4. In high throughput, SHAPE will allow examination of multiple RNAs simultaneously or the same RNA under various conditions. This unique feature makes it possible to monitor minor RNA structural response to the presence/activity of protein factors at different concentrations and test the significance of other co-factors that are important for RNA structure (e.g., divalent cations) or for protein enzymatic function (e.g., ATP). No other technology offers this unusual combination of sensitivity, flexibility for applied conditions, and high throughput. High-throughput identification of structural signatures defining oncogenic mRNAs will have tremendous potential in the discovery and design of novel, powerful anticancer drugs.
- NCI Mentor of Merit Award
Stuart Le Grice was nominated for the NCI Outstanding Mentor Award and was selected as a Mentor of Merit. Dr. Le Grice received one of the highest rankings in a competitive review in which nominees were "judged on their records as mentors; their accessibility to trainees; their ability to communicate and provide instruction and constructive feedback; their capacity to provide an environment conducive to science and learning; their propensity to give credit to trainees and promote visibility of their work; and their attention to the career development needs of those they mentor."
- Howard Temin Pathway to Independence Award (K99/R00)
Chandravanu Dash received a K99/R00 Award from the National Institute on Drug Abuse for his proposal "Role of Nucleic Acid Structure in HIV-1 Replication." The long-term goal of Dr. Dash's PI award was to elucidate the mechanism of interactions between essential viral and cellular enzymes with their nucleic acid substrates during HIV replication. New and important biochemical data obtained from this proposal would facilitate our understanding of the mechanism of HIV-1 replication, which is essential to designing better and effective drugs against HIV. Dr. Dash was mentored by Dr. Stuart Le Grice and co-mentored by Dr. Vineet KewalRamani of the HIV DRP at NCI-Frederick. He is now an Assistant Professor in the Center for AIDS Health Disparities Research at Meharry Medical College; his research program is focused on understanding how drugs of abuse influence HIV/AIDS.
- Fellows Award in Research Excellence (FARE)
Chandravanu Dasah was a FARE awardee based on his work on studying HIV RT function using nucleosidec analog mutagenersis
- Directors Innovation award
Jason Rausch was awarded $10,000 from the NCI Director's Intramural Innovation Award Program. Dr. Rausch's innovation, "Evolving Sequence-Specific Integrases and Methyltransferases by In Vitro Compartmentalization and Selection," uses a novel methodology to simultaneously screen millions of enzyme variants, with selection based both on targeted binding/activity and the absence of nonspecific binding activity. Directed evolution has never been applied in this manner to either of these enzymes, and some of the proposed methods for linking phenotype with genotype are unprecedented. Dr. Wu's innovation, "Recombinant Human Telomerase Reverse Transcriptase," makes it possible to express and purify enzymatically active recombinant human telomerase. This advance will allow a much better understanding of the structure and function of human telomerase and has the potential to be used to develop novel anticancer therapies.
- Associate Professorship – Ohio State University.
Mamuka Kvaratskhelia joined the faculty in the Pharmacy Department, Ohio State University, as an Associate Professor.