RNA binding proteins are critical for regulation of RNA function, and therefore maintaining appropriate levels of these proteins is important for cellular regulation. I focus on MAGOH, an essential subunit of the Exon Junction Complex (EJC) that is necessary for stable binding of the EJC to mRNA. Like all key regulatory proteins, MAGOH levels in the cell are tightly controlled. Reductions in MAGOH can lead to defects in embryonic development. We have found that, in human embryonic kidney (HEK293) cells, when an externally introduced copy of MAGOH (exogenous FLAG-MAGOH) is forcibly expressed from a tetracycline-inducible promoter, it causes the levels of endogenous MAGOH protein to go down. Thus, there is a feedback mechanism that maintains appropriate levels of MAGOH. The molecular details of this mechanism remain largely unknown and are the subject of my research. Here, I test if the regulation of MAGOH occurs at the transcriptional or post-transcriptional level. My results show that when increasing amounts of FLAG-MAGOH mRNA and protein are expressed in the cell, the endogenous MAGOH mRNA and pre-mRNA levels are unchanged. This data suggests that neither the degradation nor the synthesis of endogenous MAGOH mRNA is affected by more FLAG-MAGOH protein expression. Interestingly, I find that when cells are treated with a proteasome inhibitor (MG-132), the endogenous MAGOH protein does not decrease as observed in the untreated cells. Therefore, MAGOH protein levels are regulated by proteasome-mediated degradation of the protein. Additionally, the incorporation of MAGOH into the EJC seems to play a role in its regulation.No embargoAcademic Major: Molecular Genetic
