Clara Kielkopf, Ph.D.
2016 Grant Recipient
Structural mechanisms and targeting of MDS-relevant pre-mRNA splicing factors
Discovery Research Grant 2016
Patients with myelodysplastic syndromes (MDS) acquire defects in the molecular machinery for “splicing” gene transcripts into legible RNA messages. Normally, these so-called “splicing factors” process gene transcripts into legible RNA messages. We seek to understand how splicing factor mutations in turn alter gene expression in MDS, and through this information, expand the molecular toolbox for investigating and treating the disease.
We have shown that a common, S34F mutation of the U2AF1 splicing factor subtly changes its binding site preference on gene transcripts. We have used this information to identify chemicals that alter the mutated splicing factors’ ability to bind and hence “splice” gene transcripts. We have leveraged single-molecule approaches to reveal that a recurrent splicing factor mutation alters the molecular gymnastics of its RNA complex.
Future studies remain needed to determine the mechanistic implications of our new single molecule finding, as well as to explore the potential of our U2AF-targeted chemical as a means to selectively kill MDS or cancer cells, while causing less harm to normal ones.
Okeyo-Owuor, T., B.S. White, R. Chatrikhi, D.R. Mohan, S. Kim, M. Griffith, L. Ding, S. Ketkar-Kulkarni, J. Hundal, K.M. Laird, C.L. Kielkopf, T.J. Ley, M.J. Walter, T.A. Graubert, U2AF1 mutations alter sequence specificity of pre-mRNA binding and splicing Leukemia, 2015 29(4): p. 909-17 doi: 10.1038/leu.
Fei, D.L., H. Motowski, R. Chatrikhi, S. Prasad, J. Yu, S. Gao, C.L. Kielkopf, R.K. Bradley, H. Varmus, Wild-Type U2AF1 Antagonizes the Splicing Program Characteristic of U2AF1-Mutant Tumors and Is Required for Cell Survival PLOS Genet 2016, 12(10): p. e1006384 doi: 10.1371/journal.pgen.1006384
Jenkins, J.L. and C.L. Kielkopf, Splicing Factor Mutations in Myelodysplasias: Insights from Spliceosome Structures Trends Genet, 2017, 33(5): p. 336-348 doi: 10.1016/j.tig.2017.03.001
Loerch, S. and C.L. Kielkopf, Unmasking the U2AF homology motif family: a bona fide protein-protein interaction motif in disguise RNA 2016, 22(12): p. 1795-1807 doi: 10.1261/rna.057950.116
Kielkopf, C.L., Insights from structures of cancer-relevant pre-mRNA splicing factors Curr. Opin. Gen., Dev., 2018, 48:57-66 doi: 10.1016/j.gde.2017.10.008
Glasser, E., A.A. Agrawal, J.L. Jenkins, and C.L. Kielkopf, Cancer-associated mutations mapped on high resolution structures of the U2AF2 RNA recognition motifs Biochemistry 2017, 56(36): p. 4757-4761 doi: 10.1021/acs.biochem.7b00551