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Research Projects

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 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. Specific projects include:

Polypyrimidine Tract Recognition By U2AF65

Polypyrimidine Tract Recognition By U2AF<sup>65</sup>The uridine-rich sequence by the 3’ splice of the pre-mRNA is recognized by the U2 snRNP auxiliary factor large subunit, U2AF65. We have shown how U2AF65 can distinguish uridines - by specific hydrogen bonds with the base edges.

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Phosphorylation of Splicing Factor-1

Phosphorylation of Splicing Factor-1Splicing factor-1, SF1 recognizes a branch point sequence (BPS) consensus preceding the 3’ splice site and is regulated by phosphorylation. We are investigating the structural and functional consequences of phosphorylation for human SF1 interactions with U2AF65 and the pre-mRNA splice site.

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Pre-mRNA Recognition by U2AF35 Heterodimer

Pre-mRNA Recognition by U2AF<sup>35</sup> HeterodimerThe small subunit of the U2 snRNP auxiliary factor heterodimer, U2AF35, specifically contacts the 3’ splice site junction during pre-mRNA splicing. Whether U2AF35 contributes to the RNA affinity and specificity of the U2AF heterodimer, or is passively positioned to contact the RNA by U2AF65, is an outstanding question that we are currently seeking to address.

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