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.
Highlights
Of the 166 known RNA nucleoside modifications, pseudouridine (Ψ) is the most abundant post-transcriptional modification and was the first to be discovered [1,2]
Since 1.25 μg of the starting material, i.e., intact nuclear RNA is equal to 3.68 nmol, we concluded that approximately 1.20% of nucleosides in nuclear-enriched RNA are Ψs
While it is beyond the scope of our current report, we have previously shown that our Ψ quantification method can expand to include the measurement of Ψ levels in RNAs isolated through purification of specific RNP complexes, such as the
Summary
Of the 166 known RNA nucleoside modifications, pseudouridine (Ψ) is the most abundant post-transcriptional modification and was the first to be discovered [1,2]. Guided by the snoRNA, the dyskerin/NHP2/NOP10 complex carries out pseudouridylation of specific uridine residues in non-coding, as well as coding RNA [3]. The traditional physicochemical property-based chromatography method provides an alternative way to quantify relative Ψ levels by simple enzymatic digestion of cellular RNA into single nucleosides [19]. Upon separation of different nucleosides by high-performance liquid chromatography (HPLC), different detection methods have been applied to determine Ψ levels in cellular RNA pools. Absorbance detectors, such as ultraviolet (UV) detectors measuring the light absorbing capacity of the analytes, are most commonly used for HPLC analysis. We contend that the modified HPLC-UV assay provides an economic option for rapid screening of small changes in Ψ level
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