Researcher(s)
- Rebecca Payne, Biological Sciences, University of Delaware
Faculty Mentor(s)
- Jeffrey Mugridge, Chemistry and Biochemistry, University of Delaware
Abstract
The enzyme FTO belongs to the iron(II)/alpha-ketoglutarate-dependent family of dioxygenases that play a role in the regulation of gene expression and the development of metabolic diseases like cancer and obesity. FTO is an RNA demethylase that removes N6-methyladenosine (m6A) modifications on mRNA, which helps regulate mRNA transcript structure and function. Recent studies have identified the tRNA-modifying enzyme Trmt10A as an interaction partner of FTO. Trmt10A is an S-adenosylmethionine (SAM)-dependent methyltransferase that adds N1-methylguanosine (m1G) modifications to tRNAs. ZBTB48 is a C2H2-zinc finger protein that functions in relation to the maintenance of telomeres. In previous studies, it has been confirmed that FTO and both ZBTB48 and Trmt10A interact in vivo; however, the mechanism of their interaction and function has not been identified or characterized to date
A truncated version (32-505) of the FTO protein, ZBTB48 (1-298), and full-length Trmt10A (1-399) were overexpressed in E. coli BL21 cells and purified using affinity chromatography, ion exchange, and size exclusion chromatography by FPLC. SDS-PAGE was then utilized to analyze the purity of the proteins for downstream experiments. A clear interaction between FTO with either Trmt10A or ZBTB48 has not been identified so far. After purifying the intended proteins for study, a variety of pull-down assays using tagged proteins and co-elution studies by size exclusion chromatography were performed to validate and map the interactions between FTO and either Trmt10A and ZBTB48. However, addition of Trmt10A increased FTO-mediated m6A demethylation in vitro, indicating there may be possible interactions with these proteins in the presence of the substrate and cofactors. For future studies, full-length FTO constructs with Trmt10A and the full-length construct with ZBTB48 will be conducted to further map and validate these interactions.