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

ABSTRACT: Pseudouridine (?) is the most abundant post-transcriptionally modified ribonucleoside. Different ? 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 ? levels quantitatively, a previously reported high performance liquid chromatography method coupled with ultraviolet detection (HPLC-UV) was modified to determine total ? 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/?L and to have absolute ? 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 ? levels in nuclear-enriched RNA pools was observed. Our assay provides a fast and accurate quantification method to measure changes in ? levels of different RNA pools without sample derivatization.

SUBMITTER: Xu J 

PROVIDER: S-EPMC5615352 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature