ScholarWorks@UNO

Protein Interactions in RNA Methylation Complexes

Experiments were performed to test sequence and structural specific interactions of proteins with a conserved RNA modification enzyme, which is known as Ime4 in yeast and METTL3 in mammals. Ime4 methylates N6-adenosine bases on mRNA molecules. The goal of this project is to gain direct insight into how novel proteins interact with Ime4 to form the methyltranferase (MTase) complex and to identify proteins that are essential for its activity. It has been recognized that there are two proteins that interact within the Ime4 complex, which are known as Mum2 (a cytoplasmic protein essential for meiotic DNA replication within yeast) and SLZ1 (a transcription factor). We hypothesize that the N-terminal domain of Ime4 is the location of binding of the aforementioned proteins in this complex. Similarly, we are testing whether the human orthologs of Ime4 (METTL3) form an analogous complex that includes an ortholog of Mum2, known as WTAP, and its binding partner WT1. The major approaches include in vivo genetic assays in yeast to test protein-protein interactions and the use of recombinant DNA technology to construct fusion genes/deletions. The results demonstrate that Mum2 interacts with a specific, nonconserved region in the Ime4 N-terminal domain. Also, we have discovered a new binding partner, YGL036W, which interacts with the N-terminal domain of Ime4. Currently, several experiments are being carried out with the METTL3 complex and its hypothesized protein binding partners to assess the interactions of this complex. /