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

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 dysk...

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Detalles Bibliográficos
Autores principales: Xu, Jialin, Gu, Alice Y., Thumati, Naresh R., Wong, Judy M.Y.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5615352/
https://www.ncbi.nlm.nih.gov/pubmed/28872587
http://dx.doi.org/10.3390/genes8090219
Descripción
Sumario: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.