ANALYSIS OF THE MASS SILENT POST-TRANSCRIPTIONAL MODIFICATION PSEUDOURIDINE IN RNA BY MASS SPECTROMETRY
Pseudouridine has been of considerable interest in the last decade for several reasons. Of all post-transcriptional modifications found in RNA, it is the most common modification found. Because it is a structural isomer of uridine, it has the potential for more hydrogen bonding than uridine. It is found in conserved regions of RNA, therefore it may play some role in the translation process or in the ability of the ribosome to take its final, tertiary structure. Although pseudouridine has been known for quite some time, its sequence location in RNA has not been studied until recently. This is because, as a structural isomer of uridine, its analytical properties including UV absorbance, electrophoretic mobility, interaction with stationary phases and mass are similar to those of uridine. It is extremely difficult to place pseudouridine in an oligonucleotide sequence when, for all intents and purposes, it behaves as uridine. Mass spectrometric techniques alone are unable to distinguish pseudouridine. As it has the same mass as uridine, pseudouridine is a “mass-silent” modification. An interest of this lab has been to develop analytical methods to characterize pseudouridine. N-cyclohexyl-N’-?-(4-methylmorpholinium)ethylcarbodiimide selectively derivatizes pseudouridine The focus of this work is to use this selective derivatization to develop an analytical method that can determine the location of pseudouridine within an RNA, no matter the size of the RNA. This method was initially tested using E. coli . RNAs, which have been sequenced, including modifications, so it is a good organism to validate the techniques developed. Initial studies focused on tRNA, which is the smallest of all RNAs, then moved to 16S rRNA, and then finally to 23S rRNA.
School:University of Cincinnati
School Location:USA - Ohio
Source Type:Master's Thesis
Keywords:pseudouridine rrna mass spedtrometry
Date of Publication:01/01/2003