Ezekiel Delgado, a senior at the University of California, Los Angeles, characterized the RNA editing activity of deaminases using an in vitro assay.
Deaminases are a unique set of enzymes with the capacity to edit RNA and DNA. This is due to their ability to cause mutations in single nucleotide bases through the process of deamination. The Broad Summer Research Program has allowed me to grow as a researcher and most importantly as an individual. The endless support from the BSRP staff and cohort, along with the one of a kind environment that is created by all of the wonderful people at the Broad, gave me a sense of community that I have never experienced before. This allowed me to put my all into developing my research and professional skills while feeling truly supported along my own personal journey. Overall, this summer has solidified my interest in research, and provided me with essential skills to succeed in my future career.For example, the cytosine deaminase apolipoprotein B mRNA-editing enzyme 1 (Apobec1) is an RNA editing deaminase capable of modifying cytosine to uracil (C to U). Additionally the adenosine deaminases, tRNA adenosine deaminase (TadA) and adenosine deaminase acting on RNA 2 (Adar2) convert adenine to inosine (A to I). The ability to create targeted mutations in genetic material is widely applicable for a variety of applications, including gene editing for precision medicine. Previously, the Chen lab has utilized RNA deaminases for RNA timestamping, which is a method to infer the age of individual RNAs in RNA-sequencing based on the number of edits made by a deaminase on an mRNA molecule.We are interested in characterizing the RNA editing activity of a mutant TadA, Apobec1, and a mutant Adar2 in vitro for downstream application. In order to do this we expressed TadA in E. coli, and Apobec1 and Adar2 in yeast. The mutant TadA is a tRNA editing enzyme that was evolved to adopt DNA editing activity. Conversely, Apobec1 and the mutant Adar2 have RNA editing activity. After expression we then purified the proteins and evaluated their activity levels using an in vitro assay. Overall the outcomes of our experiments will inform future research by providing necessary information regarding the RNA editing activity of widely applicable deaminases.
Project: Characterizing the RNA editing activity of deaminases in vitro
Mentor: Verena Volf, Chen Lab