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Kellner Isotope Lab

RNA modifications

The central dogma of molecular biology states that DNA is the storage of the genetic code, which is transcribed into messenger RNA (mRNA) and translated into proteins with the help of transfer RNA (tRNA) and ribosomal RNA (rRNA). This fundamental life process is dominated by nucleic acids, which are composed of the canonical nucleosides adenosine, guanosine, cytidine and uridine (and thymidine in case of DNA). Additionally, RNA molecules, which are believed to be one of the first catalytic molecules in early evolutionary life, are heavily modified. Chemical variations such as methylations, thiolations or acylations or even rearrangements of the canonical building block form a second layer of information on top of the sequence code. A great overview can be found here.

Analogous to the term epigenetics, which describes the modulation of this second layer in DNA, the term epitranscriptomics was established for RNA. Our research group is focused on identifying RNA modifications and understanding their function in cell homeostasis and survival.

In yeast, it was found that tRNA modifications are highly dynamic during cellular stress and are used by the cell for efficient stress survival by changing the translational speed of stress response proteins. Although the dynamic nature of tRNA modifications is crucial to cell survival, it remains unclear how the cell achieves the adaptation of the modification profile mechanistically. Such studies are urgently needed if one considers the number of diseases related to imbalanced RNA modification profiles (Nachrichten aus der Chemie). Despite the ever increasing interest in the epitranscriptome the number of tools to study the dynamics of RNA modifications and the complex process of finding biological consequences is still limited. So far it has not been possible to distinguish between the different cellular mechanisms resulting in RNA modification such as degradation or enzymatic alteration. This is one of the challenging factors for the elucidation of the epitranscriptome.

To bypass this problem our lab uses nucleic acid isotope labeling coupled mass spectrometry, short NAIL-MS. This approach uses stable isotope labeling of RNA nucleosides, followed by RNA purification (e.g. tRNA, rRNA, mRNA) for absolute quantification by high sensitivity mass spectrometry. This enables the discrimination of RNA modification from new transcripts and modifications from already existing ones in vivo. Moreover this approach allows multiplexing of samples, the discovery of new modifications and the absolute quantification by the use of a stable isotope labeled internal standard (SILIS). Additionally, our present tools are aimed to be extended by establishing a method to analyze RNA modifications on the oligonucleotide level by a modified version of our established nucleoside based NAIL-MS method.

It is the goal of our research to extend and apply this new tool, to examine the various mechanisms and functions of RNA modification biology in various cell types and consequently shed light on the role of RNA modifications in cell homeostasis and neurological disease pathogenesis.

Topics:

  • New antibiotic targets based on novel RNA modifications
  • Stress induced RNA modification mechanisms in yeast
  • The role of RNA modifications in neurological diseases