My project was a preliminary study of the MAPPIT binary assay as a method to study protein-protein interactions (PPI) of the different ESRP1 regulated isoforms. I conducted my research in the laboratory of Dr. Russ Carstens in the Genetics Department of the Perelman School of Medicine. Using Gateway recombination technology, I worked on the positive and negative interactor pairs outlined by Braun et al. The goal of my project was to successfully use the MAPPIT system to study PPIs in mammalian cells with future plans to build a library of cDNAs encoding human ESRP1 targets compiled and transferred into relevant expression constructs.
Previous publications and work in our lab have focused on the building and “RNA maps,” where the binding position of the protein relative to the alternatively spliced exon determines inclusion or exclusion of the exon. However, our understanding of how alternative splicing events regulated by ESRPs alter function at the protein level has been limited. Given evidence that one predominant outcome of AS is to alter PPIs, this research project is part of a much larger project that aims to identify differences in PPIs of ESRP1 alternatively spliced protein isoforms.
At the Carstens lab, I learned how to apply my classroom knowledge by using a combination of techniques in my work. Moreover, I have learned how to troubleshoot and think critically about why my results may not be what I predicted. In my initial MAPPIT tests, certain positive control interactor pairs did not show protein-protein interactions. I worked systematically and carefully considered a possible reason for this unexpected outcome. After carefully revisiting the literature and running quick tests, I determined that the orientation the proteins interacted in can have an effect if one of the proteins were sterically hindered in one alignment. This research experience has taught me how to be adaptable and treat unanticipated results as a learning opportunity for investigative analysis. Therefore, while I am a person that enjoys organization and planning, my experiences with surprising outcomes have enabled me to familiarize myself with unfamiliarity and think creatively.
There was so much more to research than I had previously expected. I have come to appreciate the challenging nature of research because it makes the process of academic exploration much more rewarding.