Poster abstracts
Poster number 129 submitted by Kaley Simcox
Quantitative mapping of modified RNA nucleosides in tRNAs
Kaley M. Simcox (Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA), Joshua D. Jones (Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA), Robert T. Kennedy (Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA), Kristin S. Koutmou (Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA)
Abstract:
Transfer RNAs are the most abundantly modified RNA species with up to 20% of sites containing a modified nucleoside. RNA modifications influence all aspects of the RNA lifecycle including tRNA structure, stability, and tRNA:mRNA interactions during translation. The location of a modification on a tRNA is an important determinant of function. Therefore, rigorous methods are needed to confidently identify nucleosides and sequence tRNAs. Using a multiplexed methodology like liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) nucleoside discovery and sequencing are possible from total tRNA mixtures. Using two orthogonal enzymatic digestions, we were able to achieve high sequence coverage for E. coli total tRNA by exploiting tRNA secondary structure with single-stranded specific ribonucleases. Additionally, we have developed an untargeted LC-MS/MS method for nucleoside discovery from E. coli total tRNA. The combination of these two methodologies will be essential to elucidate the RNA modification landscape and give further insight to the role of RNA modifications.
Keywords: RNA modifications, LC-MSMS