Xu Jialin, Gu Alice Y, Thumati Naresh R, Wong Judy M Y
Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, BC V6T 1Z3, Canada.
Genes (Basel). 2017 Sep 5;8(9):219. doi: 10.3390/genes8090219.
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.
假尿苷（Ψ）是转录后修饰最为丰富的核糖核苷。不同的Ψ修饰与应激反应相关，据推测可协调细胞对多种不同应激的独特生物学反应。在不同引导RNA的帮助下，戴斯科林复合物可使核糖体RNA、小核RNA和选择性信使RNA发生假尿苷化。为了定量监测Ψ水平，对先前报道的高效液相色谱法结合紫外检测（HPLC-UV）进行了改进，以测定不同细胞RNA组分中的总Ψ水平。我们的方法经验证，在0.06 - 15.36 pmol/μL的线性范围内准确且精密，绝对Ψ定量水平低至3.07 pmol。使用我们优化的HPLC分析方法，我们发现来自HEK293细胞系的富含核RNA和小非编码RNA库中，所有核糖核苷的1.20%和1.94%，以及从HeLa细胞系分离的18S和28S rRNA中核糖核苷的1.77%和0.98%被假尿苷化。在敲低戴斯科林表达后，观察到富含核RNA库中总Ψ水平持续且显著降低。我们的分析方法提供了一种快速准确的定量方法，无需对样品进行衍生化即可测量不同RNA库中Ψ水平的变化。
