A classic view of genetic traits is that a set of defined DNA sequences are controlled simply by activation or repression of transcription. Recently RNA splicing, a process in which genes are regulated at the messenger level, has emerged as an important source of protein diversity. Most eukaryotic mRNAs (~60%) contain intervening 'junk' sequences that are removed during RNA splicing. By alternative RNA splicing of non-sequential coding regions, a single gene may encode up to thousands of variant proteins.

The primary focus of my laboratory is to investigate how RNA splice sites are recognized. The splicing machinery faces the daunting task of identifying short protein-coding regions from one another amidst a sea of intervening sequences. The essential human splicing factor U2 auxiliary factor (U2AF) Is one of the first components to recognize the 3' splice site during assembly of the splicing apparatus. In an unknown manner, U2AF forms a distinct surface that is recognized in turn by other splicing factors. As a prelude to addressing this problem, we have solved the structure of the core U2AF heterodimer. The molecular interactions of U2AF with the RNA splice site and other proteins (SAP155, URP, UAP56) are being visualized at atomic resolution utilizing the technique of X-ray crystallography. Biophysical techniques (including circular dichroism, calorimetry, RNA binding assays) and functional assays are used to characterize the protein and RNA interactions.

Relevance to Human Health. A small error in splice site selection generates aberrant proteins. Despite several levels of cellular control, the fragile nature of RNA splice site choice is illustrated by many known point mutations within consensus splice elements that are associated with human genetic disease (including types of cancer and muscular dystrophy). Our research addresses the fundamental question of how the appropriate 3' splice site is selected. In the future, we will employ structural and thermodynamic information to develop specific molecular strategies against harmful splice variants.

Biochemistry and molecular biology, gene regulation, RNA, splicing, U2AF, biophysics, structure, genetic disease

Delta Omega Honorary Public Health Society 2006 Faculty Research Initiatives Award 2005 Prostate Research Foundation Award 2005 Basil O'Connor Award 2004 Kimmel Scholar Award 2004 Faculty Innovation Grant 2003 CFAR Pilot Grant 2003 JHMRI Pilot Grant 2003 Faculty Development Award 2002 American Cancer Society Postdoctoral Fellowship 2001 National Science Foundation Graduate Fellowship 1995

Sickmier, E.A., Frato, K.E., Paranawithana, S., Shen, H. Green, M.R. and Kielkopf, C.L. (2006) Structural basis of poly-pyrimidine tract recognition by the essential splicing factor U2AF65. Molecular Cell, in press.

Sickmier, E.A., Frato K.E., and Kielkopf, C.L. (2006) Crystallization and preliminary X-ray analysis of a U2AF65 variant in complex with a polypyrimidine-tract analogue by use of protein engineering. Acta Crystallographica F62, 457-459.

Manceau, V., Swenson, M., LeCaer, J.P., Sobel, A., Kielkopf, C.L., and Maucuer, A. (2006) Major Phosphorylation of SF1 on Adjacent Ser-Pro Motifs Enhances Interaction with U2AF65. FEBS J. 273, 577-87.

Thickman, K.R., Swenson, M., Gryczynski, Z., and Kielkopf, C.L. (2006) Multiple U2AF65 binding sites within SF3b155: Thermodynamic and spectroscopic characterization of protein-protein interactions by pre-mRNA splicing factor complexes. J. Mol. Biol. 356, 664-683.

Sickmier, E.A., Brekasis, D., Paranawithana, S., Bonanno, J.B., Paget, M.S.B., Burley, S.K. and Kielkopf, C.L. (2005) X-ray Structure of a Rex-Family Repressor/NADH Complex: Insights into the Mechanism of Redox Sensing. Structure, 13, 43-54.

Kielkopf, C.L., Luecke, S. and Green, M.R. (2004) U2AF-Homology-Motifs: Protein Recognition in the RRM World, Genes & Dev. 18, 1513-1526.

Kielkopf, C.L. and Burley, S.K. (2002) X-ray structures of threonine aldolase complexes: structural basis of substrate recognition, Biochemistry, 41, 11711-11720.

Kielkopf, C.L., Rodionova, N.A., Green, M.R. and Burley, S.K. (2001) A novel peptide recognition mode revealed by the X-ray structure of a core U2AF35/U2AF65 heterodimer, Cell 106, 595–605.

Kielkopf, C.L., Erkkila, K.E., Barton, J.K. and Rees, D.C. (2000) Intercalation of a DNA Helix by a Photoexcitable Rhodium Complex at 1.2 Å Resolution, Nature Struct. Biol. 7, 117-121.

Kielkopf, C.L., Ding, S., Kuhn, P. and Rees, D.C. (2000) Conformational flexibility of B-DNA revealed at 0.74 Å Resolution: d(CCAGTACTGG), J. Mol. Biol. 296, 787-801.

Kielkopf, C.L., Bremer, R. E., White, S., Szewczyk, J., Turner, J.M., Baird, E.E., Dervan, P.B. and Rees, D.C. (2000) Structural Effects of DNA Sequence on T•A Recognition by Hydroxypyrrole/ Pyrrole Pairs in the Minor Groove, J. Mol. Biol. 295, 557-567.

Kielkopf, C.L., White, S., Szewczyk, J., Turner, J.M., Baird, E.E., Dervan, P.B. and Rees, D.C. (1998) Structural Basis for Recognition of A•T and T•A Base Pairs in the Minor Groove of B-DNA, Science. 282, 111-115.

Kielkopf, C.L., Baird, E.E., Dervan, P.B. and Rees, D.C. (1998) Structural Basis of G•C Recognition in the DNA Minor Groove, Nature Struct. Biol. 5, 104-109.

Kielkopf, C.L., Baird, E.E., Dervan, P.B. and Rees, D.C. (1998) Structural Basis of G•C Recognition in the DNA Minor Groove, Nature Struct. Biol. 5, 104-109.