Poster abstracts
Poster number 145 submitted by Pooja Madan Mohan
mRNA editing by tRNA-adenosine deaminases
Pooja Madan Mohan (Department of Biochemistry, Case Western Reserve University, Cleveland, OH 44106), Xin Lan (Department of Biochemistry, Case Western Reserve University, Cleveland, OH 44106), Yi Zhang (Department of Biochemistry, Case Western Reserve University, Cleveland, OH 44106)
Abstract:
RNA editing is a post-transcriptional modification that alters the sequence of RNA and expands the RNA landscape beyond the genetic blueprint. The most common form of RNA editing is the conversion of adenosine to inosine (A-to-I) by adenosine deaminases. In higher eukaryotes, this reaction is best characterized by double-stranded RNA–specific adenosine deaminases (ADARs), where inosine is interpreted as guanosine by the translation machinery, resulting in potential recoding of protein sequences. Besides ADARs, tRNA-specific adenosine deaminases convert adenosine at position 34 to inosine in the anticodon wobble position of tRNA, enabling codon expansion during translation, and are essential in all kingdoms of life. Despite their near-complete efficiency on editing tRNA substrates, tRNA deaminases have rarely been considered for mRNA editing because of their strong substrate selectivity toward tRNA. Our laboratory recently discovered that the essential tRNA deaminase TadA catalyzes highly efficient mRNA editing in E. coli, leading to substantial recoding of recombinant proteins, including α-synuclein. Initial experiments confirmed A-to-I editing in exogenously expressed mRNA, marked by a Tyr to Cys mutation in over 50% of the recombinant protein. To understand the requirements for substrate selection, we test TadA editing efficiency on mRNA substrates with altered nucleotide sequences to determine sequence specificity. Next, we perform gel shift assays to determine TadA-mRNA binding affinity and an in vitro deaminase assay to determine TadA catalytic efficiency. These findings will help identify the structural and positional requirements that drive TadA-mediated mRNA editing. Taking together, these results may reveal new layers of post-transcriptional gene regulation in RNA biology, expanding the current framework.
Keywords: TadA, adenosine deaminase, mRNA editing