Poster abstracts
Poster number 57 submitted by Aastha Gyawali
Simplifying eukaryotic transfer RNA Modification Mapping via FPLC
Aastha Gyawali (Department of Chemistry, University of Cincinnati), Scott Abernathy (Department of Chemistry, University of Cincinnati), Patrick Limbach (Department of Chemistry, University of Cincinnati)
Abstract:
Transfer RNA (tRNA) undergoes post-transcriptional modifications and these modified residues play important roles in RNA structure formation and stability. Moreover, tRNA modifications are known to have correlated and causal effects with a variety of human diseases. Eukaryotes can transcribe hundreds or even thousands of different tRNA sequences. Out of 150 total RNA modifications discovered, 93 modifications are found in tRNAs. Organisms typically express around 30-50 tRNA genes. However, many eukaryotic organisms may express hundreds or even thousands of different tRNA genes - even if some of these tRNAs only differ from one another by a single nucleotide change. Current LC-MS/MS approaches are unable to differentiate this complexity of tRNAs in the sample. Given the complexity of tRNA expression patterns, mass spectrometry-based RNA modification mapping, demonstrated for bacterial and archaeal organisms, can be complex. Thus, the goal of this project is to reduce the complexity of eukaryotic tRNA mixtures in a fashion that would mimic bacterial tRNAs. We selected FPLC as a fast, efficient, and reproducible fractionation method that can handle relatively large sample amounts using a strong anion exchange column with mobile phase A(20mM tris HCl, pH 8) and mobile phase B(1M NaCl + 20mM tris HCl) and weak anion exchange column with mobile phase A(20mM HEPES-KOH pH7.5) and mobile phase B (1MNaCl +20mM HEPES-KOH pH7.5). The standard sample used was 90 micrograms of total yeast tRNA for all studies. We investigated a variety of different mobile phase gradients - including both starting and ending %B - to identify those conditions that lead to appropriate and reproducible fractionation of the tRNA mixture. For each fraction, LC-MS/MS was used to identify the constituent tRNAs in each fraction, and LC-MS/MS sequencing using RNase T1 will be used in further study to verify the predictions of different yeast tRNAs present in each fraction.
Keywords: tRNA, FPLC, AEX