Production and Application of Stable Isotope-Labeled Internal Standards for RNA Modification Analysis

Abstract

Post-transcriptional RNA modifications have been found to be present in a wide variety of organisms and in different types of RNA. Nucleoside modifications are interesting due to their already known roles in translation fidelity, enzyme recognition, disease progression, and RNA stability. In addition, the abundance of modified nucleosides fluctuates based on growth phase, external stress, or possibly other factors not yet explored. With modifications ever changing, a method to determine absolute quantities for multiple nucleoside modifications is required. Here, we report metabolic isotope labeling to produce isotopically labeled internal standards in bacteria and yeast. These can be used for the quantification of 26 different modified nucleosides. We explain in detail how these internal standards are produced and show their mass spectrometric characterization. We apply our internal standards and quantify the modification content of transfer RNA (tRNA) from bacteria and various eukaryotes. We can show that the origin of the internal standard has no impact on the quantification result. Furthermore, we use our internal standard for the quantification of modified nucleosides in mouse tissue messenger RNA (mRNA), where we find different modification profiles in liver and brain tissue.

Eukaryotic life without tQCUG: the role of Elongator-dependent tRNA modifications in Dictyostelium discoideum

“…An exemplary merged chromatogram is given in Supplementary Figure S5 . For absolute quantification, calibration solutions were prepared with synthetically available nucleosides and 15 N 5 -deoxyadenosine ( 15 N 5 -dA) was used as an internal standard ( 57 ).…”

“…10 μL of each sample was injected to LC–MS/MS analysis (corresponding to around 150 ng tRNA digest). Note that hypermodified uridine nucleosides are unstable after digestion ( 57 ).…”

Cell culture NAIL-MS allows insight into human RNA modification dynamicsin vivo

“…In a first step, we purified tRNA Phe GAA from HEK 293 cells using a complementary DNA probe 13 . We used our established isotope dilution LC-MS/MS analysis for absolute quantification of modified nucleosides and plotted the modification profile in Figure 1 16 . For pseudouridine (Ψ), dihydrouridine (D), 2dimethylguanosine (m 22 G) and 2'-O-methylguanosine (Gm) our experimental data matches the expected values and we see full modification 32 .…”

“…One aliquot (2/3 Vol) was used for immediate RNA isolation and purification, while the remaining aliquot of the labeled and unlabeled cells were mixed and RNA was co-isolated and co-purified ( Figure 3a and Figure S13). The total tRNA was enzymatically digested to nucleosides and their abundance determined by isotope dilution mass spectrometry 16 . In the aliquot from unlabeled samples, only unlabeled nucleosides were detectable, while the aliquot of the labeled cells showed mainly signals (>98%) for labeled nucleosides.…”

“…After incubation for 2 h at 37 °C, 20 µL of LC-MS buffer A (QQQ) was added to the mixture and then filtered through 96-well filter plates (AcroPrep Advance 350 10 K Omega, PALL Corporation, New York, USA) at 3000 ×g and 4 °C for 30 min. A stable isotope labeled internal standard (SILIS) was produced in S. cerevisiae using 13 C and 15 N rich growth medium (Silantes, Munich, Germany, Product# 111601402) following recently described procedures 16,31 . 1/10 Vol.…”