The Faculty

Focus

Chemistry of RNA splicing and catalysis

Biography

Born Wilkes-Barre, Pennsylvania, 1960.
University of Scranton, B.Sc., 1982.
Rheinisch Westfalische Technische Hochschule Aachen, Fulbright Scholar, 1983.
Harvard University, Ph.D., 1989.
Harvard Traveling Scholar, Swiss Federal Institute of Technology, 1986-1989.
University of Colorado at Boulder, Howard Hughes Postdoctoral Research Fellow, 1989-1993.
The University of Chicago, Assistant Professor, 1993-2000.
Assistant Investigator, Howard Hughes Medical Institute, 1994-2000.
The University of Chicago, Professor, 2000-.
Associate Investigator, Howard Hughes Medical Institute, 2000-2004.
Investigator, Howard Hughes Medical Institute, 2004-.
Joint Appointment in the Department of Biochemistry and Molecular Biology.

Research Interests

Our group is broadly interested in the chemistry and biochemistryof nucleic acids with particular emphasis onRNA and RNA catalysis. The laboratory integrates areasof organic chemistry, physical chemistry, enzymologyand molecular biology to gain a fundamental understandingof nucleic acid structure and mechanisms ofRNA catalysis. Using the principles and techniques oforganic chemistry and molecular biology, we manipulatethe structure of RNA molecules at precise locations inways that are designed to answer very specific questionsabout biological function.

Mechanism of RNA Catalysis
We employ these approaches toward gaining a fundamentalunderstanding of the role that divalent metal ionsplay in phosphoryl transfer reactions that occur duringRNA splicing, a fundamental step in genetic expression.One experimental system that we are using to addressthese issues is the self-splicing intervening sequenceRNA of the ciliated protozoan Tetrahymena. Shortenedforms of this RNA can act as enzymes, catalyzing thesequence specific cleavage of RNA and DNA substrateswith multiple turnover. We have used sulfur substitutionof the oxygen substituents on the phosphoryl groupundergoing transfer to reveal the transition state interactionsbetween the ribozyme and the scissile phosphate.Another area of interest is the development of new methodsand model systems for studying RNA molecules. Forexample, we have recently designed a series of nucleosideanalogues, in which the C2Õ-beta hydrogen atom ofthe ribose is replaced by CH3, CH2F, CHF2, or CF3.These analogues provide a systematic way to perturb theacidity of the 2'-OH group, thereby allowing us to probethe all important role of this functional group in RNAmediated biological processes.

RNA-Protein Interactions
Restrictocin is a small protein (149 amino acids) that isso toxic that a single molecule can kill an entire cell. Thisprotein from Aspergillus restrictus is a member of a groupof functionally homologous cytotoxins, which includesthe better-known sarcin, and the mechanism of toxicity isfascinating. The single protein is able to cross the cellmembrane and cleave the 23Ð28S ribosomal RNA at asingle phosphodiester bond. The cleavage site resides ina region of the ribosomal RNA known as the sarcin/ricinloop (SRL), which folds into a tetraloop motif and abulged-G motif. The SRL participates in the binding ofelongation factors during protein synthesis. Consideringthat the 28S ribosomal RNA contains thousands of phosphodiesterbonds, the apparent specificity of this ribonucleaseis remarkable. This single cleavage event inactivatesthe ribosome and consequently abolishes its abilityto carry out protein synthesis, which ultimately leadsto death of the cell.

This scenario immediately prompts a number of questions:How does the protein cross the cell membrane?Does it really possess the attributed specificity? Is everyribosome in the cell inactivated or does a single inactivationevent lead to activation of an apoptotic pathway?Additionally, the potency of this protein immediatelysuggests a potential clinic use as an anticancer drug. Allof these are interesting questions that we hope to answer.In addition, this system has broader significance in biologyas a model system to study RNA-protein interactions,which are ubiquitous and mediate numerous importantevents during gene expression. The crystal structures ofrestrictocin and the SRL RNA have been solved in isolation,and Carl CorrellÕs lab (University of Chicago) hassolved a structure of an SRL analog in complex withrestrictocin. Upon complex formation the geometry ofthe tetraloop is dramatically rearranged by base restackingand base flipping. Remarkably, few functional studieshave been reported on this protein. Our initial focus willbe to determine the dynamic changes that occur in theSRL when it binds to restrictocin and to elucidate theenergetic contributions that enzyme-RNA substrate contactsplay in cleavage-site recognition and catalysis.

Selected References

Frederiksen JK, Li NS, Das R, Herschlag D, Piccirilli JA. Metal-ion rescue revisited: Biochemical detection of site-bound metal ions important for RNA folding. RNA. 2012;18(6):1123-41. PMCID: 3358636.

Sengupta RN, Herschlag D, Piccirilli JA. Thermodynamic evidence for negative charge stabilization by a catalytic metal ion within an RNA active site. ACS Chem Biol. 2012;7(2):294-9. PMID: 22029738

Wong KY, Gu H, Zhang S, Piccirilli JA, Harris ME, York DM. Characterization of the reaction path and transition states for RNA transphosphorylation models from theory and experiment. Angew Chem Int Ed Engl. 2012;51(3):647-51. PMCID: PMC3448066

Forconi M, Schwans JP, Porecha RH, Sengupta RN, Piccirilli JA, Herschlag D. 2'-Fluoro substituents can mimic native 2'-hydroxyls within structured RNA. Chem Biol. 2011;18(8):949-54. PMCID: 3167488.

Forconi M, Porecha RH, Piccirilli JA, Herschlag D. Tightening of active site interactions en route to the transition state revealed by single-atom substitution in the guanosine-binding site of the Tetrahymena group I ribozyme. J Am Chem Soc. 2011;133(20):7791-800. PMCID: 3119543.

Koldobskaya Y, Duguid EM, Shechner DM, Suslov NB, Ye J, Sidhu SS, et al. A portable RNA sequence whose recognition by a synthetic antibody facilitates structural determination. Nat Struct Mol Biol. 2011;18(1):100-6. PMCID: 3058332.AS

Harris ME, Dai Q, Gu H, Kellerman DL, Piccirilli JA, Anderson VE. Kinetic isotope effects for RNA cleavage by 2'-O- transphosphorylation: nucleophilic activation by specific base. J Am Chem Soc. 2010;132(33):11613-21. PMCID: 2946848.

Forconi M, Sengupta RN, Piccirilli JA, Herschlag D. A rearrangement of the guanosine-binding site establishes an extended network of functional interactions in the Tetrahymena group I ribozyme active site. Biochemistry. 2010;49(12):2753-62. PMCID: 2860537.

Forconi M, Sengupta RN, Liu M-C, Sartorelli AC, Piccirilli JA, Herschlag D. Structure and function converge to identify a hydrogen bond in a group I ribozyme active site. Angew Chem Int Ed Engl. 2009;48(39):7171-5. PMCID: PMC2862986

Forconi M, Lee J, Lee JK, Piccirilli JA, Herschlag D. Functional identification of ligands for a catalytic metal ion in group I introns. Biochemistry. 2008;47(26):6883-94. PMCID: 2758101.

Forconi M, Piccirilli JA, Herschlag D. Modulation of individual steps in group I intron catalysis by a peripheral metal ion. RNA. 2007;13(10):1656-67. PMCID: 1986806.

Hougland JL, Kravchuk AV, Herschlag D, Piccirilli JA. Functional identification of catalytic metal ion binding sites within RNA. PLoS Biol. 2005;3(9):e277. PMCID: 1184590.

Das SR, Piccirilli JA. General acid catalysis by the hepatitis delta virus ribozyme. Nat Chem Biol. 2005;1(1):45-52. PMID: 16407993

Shan S, Kravchuk AV, Piccirilli JA, Herschlag D. Defining the catalytic metal ion interactions in the Tetrahymena ribozyme reaction. Biochemistry. 2001;40(17):5161-71 PMID: 11318638.

Kuo LY, Piccirilli JA. Leaving group stabilization by metal ion coordination and hydrogen bond donation is an evolutionarily conserved feature of group I introns. Biochim Biophys Acta. 2001;1522(3):158-66. PMID: 11779630