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Pre-mRNA Splicing for Treatment of Human Disease

Illustration of RNA binding sites used by lab

RNA Spice Site Recognition by Key Proteins

Human life depends on pre-mRNA splicing for cellular viability, differentiation and responses to changing physiology or environment. A major focus of my laboratory is to understand at a molecular level how the splicing machinery identifies sites for excision from gene transcript RNAs, which in turn changes the proteins produced. We have characterized the three-dimensional shapes of human splicing proteins recognizing one another and the gene transcript RNA at high resolution by X-ray crystallography (e.g. diagram above) complemented by molecular biology in human cells. Through this research, we identify a network of interactions responsible for recognizing human splice sites. The broader impact of this work for human disease is emphasized by the severe defects in pre-mRNA splicing that accompany most human hematologic malignancies and many metabolic disorders, as well as the dependence of HIV-1 and other complex retroviruses on RNA splicing for infectivity. Ongoing research projects include:

Molecular Recognition During pre-mRNA Splicing (NIH R01 GM070503)

The overall goal of this research is to understand how essential ternary complexes of SF1, U2AF65, and U2AF35 splicing factors recognizes 3´ splice sites and initiates the process of spliceosome assembly. Specifically, we will: (i) Determine the three-dimensional shape of the full assembly of human splicing proteins bound to the gene transcript; (ii) Understand the function of splicing factor phosphorylation and its potential to serve as a drug target; (iii) Leverage emerging views of splice site signal recognition as a basis to predictably target and correct defective splice sites. The proposed research will show the molecular underpinnings of 3´ splice site selection, and thus have ramifications for understanding and potentially correcting pre-mRNA splicing defects in human diseases.

Molecular Actions of Prevalent U2AF1 Mutations in Myelodysplastic Syndromes (Edward P. Evans Foundation)

Specific mutations in the U2AF1 proto-oncogene, which encodes the U2AF35 protein, are prevalent among patients with hematological malignancies, including 10-12% of patients with myelodysplastic syndrome (MDS) without ring sideroblasts and 8-11% of patients with chronic myelomonocytic leukemia (CMML). The major goals of this project are to investigate the molecular and structural mechanisms for altered pre-mRNA splicing in MDS patients carrying somatic mutations of the U2AF1 pre-mRNA splicing factor. Specifically, we will: (i) Determine the effect of prevalent U2AF1 mutations on U2AF35 binding affected pre-mRNA splice sites of MDS patient samples, focusing on known proto-oncogenic transcripts; (ii) Determine structures of the U2AF35 protein bound to splice RNA and evaluate the locations of the mutated residues; (iii) Screen chemical libraries for inhibitors of mutant U2AF35 complexes. The results of these aims provide a foundation for understanding the molecular and structural roles of U2AF1 in aberrant pre-mRNA splicing that leads to MDS and other malignancies. This approach will also identify selective modulators of U2AF1 mutants that can provide potential long-term therapies in a new genomic age of Precision Medicine.

Structural Control of Human Co-factors for Retroviral Gene Expression (NIH R01 GM117005)

Viruses such as HIV-1 use the human machinery to produce its RNA transcripts for protein expression and ultimately genomic replication. How viruses hijack cellular processes through interactions with host macromolecules is a fundamental question in biology and medicine. In this project, we investigate the molecular mechanisms for a human splicing factor, Tat-SF1 to coordinate production of Human Immunodeficiency Virus type 1 (HIV-1) RNAs. Specifically, we will determine and test biochemical and structural roles for Tat-SF1 interfaces with: (i) the human U2 small nuclear RNA of the splicesome; (ii) the SF3b155 subunit of the U2 snRNP; and (iii) HIV-1 for co-opting of the host splicing machinery. Targeting host cofactors offers an alternative strategy to develop new anti-HIV therapeutics. Results of these experiments will lay groundwork for understanding the interplay of a key host and viral process, and potential new avenues for therapeutic development.