Poster abstracts
Poster number 65 submitted by Priti Thakur
Identification of the amino acids associated with the novel ribonuclease activity of Cusativin via protein engineering and LC-MS
Priti Thakur (Rieveschl Laboratories for Mass spectrometry, University of Cincinnati, Cincinnati OH), Patrick A. Limbach (Rieveschl Laboratories for Mass spectrometry, University of Cincinnati, Cincinnati OH), Balasubrahmanyam Addepalli (Rieveschl Laboratories for Mass spectrometry, University of Cincinnati, Cincinnati OH)
Abstract:
Cytidine specific ribonuclease (RNase), cusativin, is a useful biochemical tool in Mass spectrometry (MS)-based RNA modification mapping [1-2]. This RNase has a novel characteristic of not cleaving bonds between two adjacent cytidines [3]. This characteristic makes it even more useful since it does not generate single nucleotides, instead, generate longer digestion products that would help match to genomic sequence and increase the sequence coverage. This unique feature of cusativin has encouraged us to identify the key amino acid residues associated with this unique property. Through protein engineering involving site-directed mutagenesis, and liquid chromatography coupled with tandem mass-spectrometry (LC-MS) analysis, we are identifying the key amino acid residues responsible for this behavior. We observed changes in base recognition behavior of mutants following analysis of digestion products of synthetic RNA and yeast tRNAPhe substrates. Our preliminary experimental data shows that more than two amino acid residues are responsible for absence of CpC bond cleavage by cusativin. Implications of these findings and utility for RNA modification mapping will be discussed.
References:
1. Kowalak J, et al. (1993) Nucleic Acid Research, 21(19): 4577–85.
2. Thakur P et al (2020) Analyst. 145(3):816-827.
2. Addepalli B, et al. (2017) Anal Bioanal Chem, 409:5645–5654.
Keywords: Ribonuclease, Cusativin, mass-spectrometry