Poster abstracts

Poster number 47 submitted by Anita Durairaj

Identifying pseudouridines in RNA by mass spectrometry

Anita Durairaj (Chemistry, University of Cincinnati), Patrick A. Limbach (Chemistry, University of Cincinnati)

Abstract:
Pseudouridine is the most abundant and common posttranscriptional modification found in RNA. Pseudouridines are found at specific locations in the rRNAs and tRNAs of Archaea, Bacteria and Eukarya. However the function of pseudouridines on a molecular level remains less understood. The localization and identification of pseudouridines in RNA is required in order to decipher their functional role. Appropriate experimental techniques have been developed for the identification of pseudouridines but these are often labor-intensive and time and sample-consuming.

In this work, we describe a mass spectrometry-based approach for the detection of pseudouridines in RNA. In our approach, the RNA is first digested into fragments by the endonucleases, RNase A or T1. Mass spectral data of the digest fragments are obtained and serve as the control. The digest products are then subsequently derivatized with 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide (CMC). Pseudouridines are identified by a direct comparison of the control (non-derivatized digest fragments) to their CMC-derivatized counterparts. After CMC derivatization, pseudouridine residues exhibit a mass shift of 252 Da that allows its presence to be easily detected by mass spectrometry.

Here, we demonstrate the application of this approach to pseudouridine identification in a mixture of Escherichia coli tRNAs. Current efforts are focused towards the detection of pseudouridines in larger RNAs, such as the 23S rRNA of E. coli. We find that this approach enables the rapid screening for pseudouridines in both small and large RNA systems.

Keywords: pseudouridine, mass spectrometry, endonuclease