Applying Silantes Cell Growth Media to study Human RNA Modification Dynamics

Nucleic acids play a critical part in Transcription and Translation, which are fundamental processes for life on earth. They are the building blocks of DNA, which represents the genetic code that is transcribed into messenger RNA (mRNA) and then translated into proteins, with the help of transfer RNA (tRNA) and ribosomal RNA (rRNA).

The study of RNA modification dynamics holds great potential for elucidating disease pathogenesis. In this regard RNA modifications have been shown to be involved in cancer, neuronal diseases and mitochondrial diseases. For a number of reasons the study of cellular mechanisms that involve RNA modifications are challenging. One approach that has great potential to overcome these challenges is the use of nucleic acid isotope labeling coupled with mass spectrometry (NAIL-MS).

Article: https://kaiser-lab.de/research/rna-modifications/

Using NAIL-MS to analyze human RNA modifications in-vivo

The Kaiser lab at Goethe University in Frankfurt uses isotope labeled yeast cells that were cultivated in rich growth media from Silantes to trace human RNA modification dynamics in-vivo. According to Prof. Kaiser, this requires the analysis of nucleic acids by mass spectrometry.

For the quantification of the numerous RNA modifications an internal standard is required that differs from the analyte in mass, while having nearly the same physicochemical properties. This ensures that the internal standard co-elutes with the analyte, but is distinguished from the analyte due to the different m/z value.

The team of Prof. Kaiser produces this internal standard biosynthetically. For this purpose, yeast is cultivated in ¹³C- and ¹⁵N-labeled rich growth media from Silantes. The yeast then incorporates the label and the generated isotopologues are then added to the samples to be studied.

Prof. Kaiser stated that “Complete isotopic labeling of all nucleosides was only achieved by using the rich growth media from Silantes.”

The cell culture NAIL-MS method developed by Prof. Kaiser allows insights into human tRNA/rRNA modification dynamics in-vivo by feeding the model organism with stable isotopologues as nutrients. The resulting isotope labeled nucleic acids are digested into nucleosides and then analyzed by triple quadrupole mass spectrometry. In an example study, HEK293 cells were fed with isotopically labeled adenine and uridine.

The Kaiser Method

Prof. Kaiser method works as follows: ¹³C and ¹⁵N rich growth medium is generated with 1% ¹³C₆-glucose, then inoculated with a single colony S. cerevisiae and cultivated overnight. In order to reach a complete isotope labeling the culture is brought to an OD of 0.1 by adding fresh ¹³C and ¹⁵N rich growth medium that was enriched with ¹³C₆ -glucose. The cultivation is continued for two days. Subsequently, the nucleic acids can be isolated and used for the absolute quantification of samples.

According to Prof. Kaiser, complete isotopic labeling is critical if a biosynthetic stable isotope labeled internal standard (SILIS) is used. Complete labeling of all nucleosides was achieved with the Silantes ¹³C and ¹⁵N rich growth medium.

Article: https://kaiser-lab.de/research/stress-induced-rna-modification-mechanisms-in-yeast/