NSF Award
1708732
With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Dr. Sarah Michel from the University of Maryland Baltimore to investigate how the trace metal zinc is involved in the important biological process of RNA regulation. All living systems require RNA to be processed into different molecular forms. Genome sequencing has identified a family of proteins called 'non-classical' zinc finger (ZF) proteins that play key roles in regulating RNA. Two proteins from this family, TTP and CPS30, are investigated in vitro and in cells to determine how zinc binding affects RNA processing. These studies help define general principles of metal directed protein RNA regulation in living systems and identify how toxic metals disrupt these fundamental biological processes. Graduate student participants receive training at the interface of chemistry and biology using cutting edge experimental techniques. A successful outreach program called SIMSI developed by the PI with a women's college in Baltimore, which provides mini-graduate school experiences and mentoring continues. In addition, a new outreach workshop called "Minding Your Metals" that draws inspiration form the Flint, MI drinking water crisis is developed for underrepresented minority (URM) students from three Baltimore City urban public middle schools. Graduate students are involved in all outreach efforts, and will be trained in communicating findings and conveying scientific principles to audiences with different levels of science knowledge. <br/><br/>This proposal is focused on the CCCH class of non-classical zinc finger (ZF) proteins. Two CCCH ZFs, tristetraprolin (TTP) and cleavage and polyadenylation specificity factor 30 (CPSF30) are investigated. TTP is a key player in the regulation of inflammation, when reactive oxygen species are elevated, and outstanding questions include the roles of metal ions (native and toxic), and the participation of reactive oxygen species and reactive sulfur species in the structure and function of TTP. CPSF30 is involved in pre- mRNA processing, and contains a 2Fe-2S cluster, in addition to zinc sites. The PI (1) determines how metal ions, reactive oxygen species and reactive sulfur species modulate TTP function in vitro and in cells and (2) determines the mechanism of RNA recognition for CPSF30 by deciphering the functional roles of the 2Fe-2S cluster, the CCCH domains and the CCHC domain. Biochemical, biophysical, structural and cell based approaches are taken, including fluorescence anisotropy to measure protein/RNA binding, native ESI-MS and ICP-MS to determine stoichiometry, cryo-ESI-MS to elucidate reactivity with reactive oxygen and sulfur species, HDX/MS and crosslinking/ESI-MS to identify structural changes upon RNA binding, and cellular reporter assays to translate in vitro findings to cells.
