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Genes 2017, 8(9), 219; doi:10.3390/genes8090219

Quantification of Pseudouridine Levels in Cellular RNA Pools with a Modified HPLC-UV Assay

Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, BC V6T 1Z3, Canada
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Academic Editor: Kumiko Ui-Tei
Received: 25 July 2017 / Revised: 28 August 2017 / Accepted: 30 August 2017 / Published: 5 September 2017
(This article belongs to the Special Issue RNA Modification)
View Full-Text   |   Download PDF [1033 KB, uploaded 5 September 2017]   |  

Abstract

Pseudouridine (Ψ) is the most abundant post-transcriptionally modified ribonucleoside. Different Ψ modifications correlate with stress responses and are postulated to coordinate the distinct biological responses to a diverse panel of cellular stresses. With the help of different guide RNAs, the dyskerin complex pseudouridylates ribosomal RNA, small nuclear RNA and selective messenger RNAs. To monitor Ψ levels quantitatively, a previously reported high performance liquid chromatography method coupled with ultraviolet detection (HPLC-UV) was modified to determine total Ψ levels in different cellular RNA fractions. Our method was validated to be accurate and precise within the linear range of 0.06–15.36 pmol/μL and to have absolute Ψ quantification levels as low as 3.07 pmol. Using our optimized HPLC assay, we found that 1.20% and 1.94% of all ribonucleosides in nuclear-enriched RNA and small non-coding RNA pools from the HEK293 cell line, and 1.77% and 0.98% of ribonucleosides in 18S and 28S rRNA isolated from the HeLa cell line, were pseudouridylated. Upon knockdown of dyskerin expression, a consistent and significant reduction in total Ψ levels in nuclear-enriched RNA pools was observed. Our assay provides a fast and accurate quantification method to measure changes in Ψ levels of different RNA pools without sample derivatization. View Full-Text
Keywords: pseudouridine; dyskerin; HPLC; RNA modification pseudouridine; dyskerin; HPLC; RNA modification
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Xu, J.; Gu, A.Y.; Thumati, N.R.; Wong, J.M. Quantification of Pseudouridine Levels in Cellular RNA Pools with a Modified HPLC-UV Assay. Genes 2017, 8, 219.

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