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Assistant Professor of Biochemistry and Biophysics
Ph.D. California Institute of Technology 1999

Structural and Biophysical Basis of Normal and Disease-Associated pre-mRNA Recognition.

Up to half of human genetic diseases, including cancers and neuromuscular disorders, are associated with defects in splicing of pre-mRNA transcripts. Almost all human genes contain intervening, non-coding sequences that must be spliced from the pre-mRNA transcripts before translation into proteins. The majority of these are alternatively spliced to encode a number of protein variants depending on different cellular needs. My laboratory uses structural and biophysical techniques to understand how pre-mRNA splice sites are recognized in normal cells, and to suggest possible means for treating defective pre-mRNA splicing observed in human genetic diseases.

The major focus of the laboratory is the mechanism of 3’ splice site recognition by an essential pre-mRNA splicing factor, U2 Auxiliary Factor (U2AF). Using biophysical methods including X-ray crystallography, fluorescence anisotropy, calorimetry, and surface plasmon resonance, we characterize the three-dimensional shapes and energetic forces that enable U2AF to recognize the pre-mRNA splice site. We determined the X-ray structure of U2AF bound to a polyuridine sequence. Now, we trying to understand how U2AF adapts to a variety of natural RNA sequences. A long term goal of the laboratory is to determine structures of higher order 3’ splice site complexes. Knowledge of the key interactions between splicing factors and the pre-mRNA will assist development of chemotherapeutics targeted at the level of pre-mRNA splicing.

Structure-based model and schematic diagram of splicing factors (U2AF subunits and SF1) bound to the 3' splice site of the pre-mRNA.

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Recent Publications

Manceau V, Kielkopf CL, Sobel A, Maucuer A (2008) Different requirements of the kinase and UHM domains of KIS for its nuclear localization and binding to splicing factors. J Mol Biol, 381:748-62

Jenkins JL, Shen H, Green MR, Kielkopf CL (2008) Solution Conformation and Thermodynamic Characteristics of RNA Binding by the Splicing Factor U2AF65. J Biol Chem, 283:33641-9

Lin Y, Kielkopf CL (2008) X-ray structures of U2 snRNA-branchpoint duplexes containing conserved pseudouridines. Biochemistry, 47:5503-14

Kumar AO, Swenson MC, Benning MM, Kielkopf CL (2008) Structure of the central RNA recognition motif of human TIA-1 at 1.95A resolution. Biochem Biophys Res Commun, 367:813-9

Thickman KR, Sickmier EA, Kielkopf CL (2007) Alternative conformations at the RNA-binding surface of the N-terminal U2AF(65) RNA recognition motif. J Mol Biol, 366:703-10

Swenson MC, Paranawithana SR, Miller PS, Kielkopf CL (2007) Structure of a DNA repair substrate containing an alkyl interstrand cross-link at 1.65 A resolution. Biochemistry, 46:4545-53

Sickmier EA, Frato KE, Kielkopf CL (2006) Crystallization and preliminary X-ray analysis of a U2AF65 variant in complex with a polypyrimidine-tract analogue by use of protein engineering. Acta Crystallograph Sect F Struct Biol Cryst Commun, 62:457-9

Sickmier EA, Frato KE, Shen H, Paranawithana SR, Green MR, Kielkopf CL (2006) Structural basis for polypyrimidine tract recognition by the essential pre-mRNA splicing factor U2AF65. Mol Cell, 23:49-59

Thickman KR, Swenson MC, Kabogo JM, Gryczynski Z, Kielkopf CL (2006) Multiple U2AF65 binding sites within SF3b155: thermodynamic and spectroscopic characterization of protein-protein interactions among pre-mRNA splicing factors. J Mol Biol, 356:664-83

Manceau V, Swenson M, Le Caer JP, Sobel A, Kielkopf CL, Maucuer A (2006) Major phosphorylation of SF1 on adjacent Ser-Pro motifs enhances interaction with U2AF65. Febs J, 273:577-87

Sickmier EA, Brekasis D, Paranawithana S, Bonanno JB, Paget MS, Burley SK, Kielkopf CL (2005) X-ray structure of a Rex-family repressor/NADH complex insights into the mechanism of redox sensing. Structure, 13:43-54

Kielkopf CL, Lucke S, Green MR (2004) U2AF homology motifs: protein recognition in the RRM world. Genes Dev, 18:1513-26

Kielkopf CL, Rodionova NA, Green MR, Burley SK (2001) A novel peptide recognition mode revealed by the X-ray structure of a core U2AF35/U2AF65 heterodimer. Cell, 106:595-605

Kielkopf CL, Ding S, Kuhn P, Rees DC (2000) Conformational flexibility of B-DNA at 0.74 A resolution: d(CCAGTACTGG)(2). J Mol Biol, 296:787-801

Kielkopf CL, Erkkila KE, Hudson BP, Barton JK, Rees DC (2000) Structure of a photoactive rhodium complex intercalated into DNA. Nat Struct Biol, 7:117-21

Kielkopf CL, White S, Szewczyk JW, Turner JM, Baird EE, Dervan PB, Rees DC (1998) A structural basis for recognition of A.T and T.A base pairs in the minor groove of B-DNA. Science, 282:111-5

Kielkopf CL, Baird EE, Dervan PB, Rees DC (1998) Structural basis for G.C recognition in the DNA minor groove. Nat Struct Biol, 5:104-9

Graduate students in my laboratory work toward a Ph.D. degree in the following program[s]:

Ph.D. in Biochemistry
Ph.D. in Biophysics

Ph.D. candidates in my laboratory may also be affiliated with these programs:

Contact Information

E-Mail: clara_kielkopf@urmc.rochester.edu

Clara L. Kielkopf
Department of Biochemistry and Biophysics
University of Rochester School of Medicine and Dentistry
601 Elmwood Ave, Box 712
Rochester, New York 14642

Office: Medical Center 3-8546
Telephone: (585)-273-4799; Fax: (585) 275-6007