Alumni

Research interest:

– tRNA and mRNA modification

– Bacteria and Yeast

– Human cells and autophagy

Research interest:

– Mass spectrometry of proteins

– Mass spectrometry of RNA

PhD start date: 1. November 2016

PhD end date: 04. September 2020

Topic: Biosynthetic nucleic acid isotope labeling

In bacteria we can label RNA with the isotopes 15N, 34S and 13C for total labeling and deuterium in methyl groups (CD3). With this isotope labeling in combination with
LC-MS/MS we can identify the sum formula and solve the structure of unknown modifications. We can do enzymatic studies in order to find out if modifications are added to the RNA by accident or enzymatically and we developed strategies to follow modifications over time in pulse chase experiments. For stress initiation we use different compounds, e.g. alkylating agents or antibiotic compounds. Afterwards we look for changed modification patterns in different RNAs and if the RNA was damaged by the stress, we follow the repair and enzyme requisition.

Publications

• Borek, C.#, Reichle, V.F.# and Kellner, S. (2020) Synthesis and metabolic fate of 4‐methylthiouridine in bacterial tRNA. # shared 1st author accepted at ChemBioChem
• Reichle, V.F., Petrov, D.P., Weber, V., Jung, K. and Kellner, S. (2019) NAIL-MS reveals the repair of 2-methylthiocytidine by AlkB in E. coli. Nat Commun, 10, 5600. PMID: 31811240
• Reichle V., Weber V. and Kellner S. (2018) NAIL-MS in E. coli determines the source and fate of methylation in tRNA , ChemBioChem, PMID: 30328661
• Reichle VF, Kaiser S, Heiss M, Hagelskamp F, Borland K, Kellner S. (2018) Surpassing limits of static RNA modification analysis with dynamic NAIL-MS, Methods, PMID: 30395967
• Heiss, M., Reichle, V. & Kellner, S. (2017). Observing the fate of tRNA and its modifications by nucleic acid isotope labeling mass spectrometry: NAIL-MS. RNA Biol. 2017 May 10:0. doi: 10.1080/15476286.2017.1325063.

September 2016-March 2017: Master Thesis on the dynamics of RNA modifications in S. cerevisiae.

Since June 2017: PhD thesis on the dynamics of RNA modifications in neurological disease.

PhD end: 09.07.2021

Investigation of RNA modification dynamics in neurological disease by NAIL-MS

RNA modifications are involved in many molecular mechanisms such as translation fidelity or folding of tertiary structures. Deviations of the “normal” modification pattern inside a cell most often results in diverse neurological diseases.

So far, it was challenging to investigate these modification dynamics, as most methods only display a static view. With our developed NAIL-MS approach (nucleic acid isotope labeling coupled mass spectrometry) we want to get a deeper insight into modification dynamics in several organisms.

I am particularly interested in the investigation of modification dynamics in human cell culture. Knockout strains or multiple stressors are used to see how the cells react to environmental challenges. Therefore I use NAIL-MS: The general workflow consist of heavy isotope labeling of RNA nucleosides by feeding precursor molecules of nucleoside biosynthesis. After total RNA isolation and purification of the desired RNA species by size exclusion chromatography (e.g. tRNA, rRNA), the RNA is digested to nucleosides and finally analyzed by high sensitivity mass spectrometry. This allows the discrimination of isotopomers and thereby enables the elucidation of many RNA modification mechanisms inside the cell.

Publications

• Thumbs, P. et al., Synthesis of Galactosylqueuosine and Distribution of Hypermodified Q-nucleosides in mouse tissues, accepted at Angewandte Chemie.
• Barraud, P., Gato, A., Heiss, M., Catala, M., Kellner, S. and Tisne, C. (2019) Time-resolved NMR monitoring of tRNA maturation. Nat Commun, 10, 3373. PMID: 31358763
• Gkatza, N.A., Castro, C., Harvey, R.F., Heiss, M., Popis, M.C., Blanco, S., Bornelov, S., Sajini, A.A., Gleeson, J.G., Griffin, J.L. et al. (2019) Cytosine-5 RNA methylation links protein synthesis to cell metabolism. PLoS Biol, 17, e3000297. PMID: 31199786
• Reichle VF, Kaiser S, Heiss M, Hagelskamp F, Borland K, Kellner S. (2018) Surpassing limits of static RNA modification analysis with dynamic NAIL-MS, Methods, PMID: 30395967
• Heiss, M., Reichle, V. & Kellner, S. (2017). Observing the fate of tRNA and its modifications by nucleic acid isotope labeling mass spectrometry: NAIL-MS. RNA Biol. 2017 May 10:0. doi: 10.1080/15476286.2017.1325063.
• Heiss, M., & Kellner, S. (2016). Detection of nucleic acid modifications by chemical reagents. RNA Biol. 2016 Nov 30:1-9. doi: 10.1080/15476286.2016.1261788.

Topic: Analytical tools to study RNA writers and erasers in vitro

Publications

• Hagelskamp, F., Borland, K., Ramos, J., Hendrick, A., Fu, D. and Kellner S. (2020) Broadly applicable oligonucleotide mass spectrometry for the analysis of RNA writers and erasers in vitro. Nucleic Acids Res. PMID: 32083657
• Ramos, J., Han, L., Li, Y., Hagelskamp, F., Kellner, S.M., Alkuraya, F.S., Phizicky, E.M. and Fu, D. (2019) Formation of tRNA wobble inosine in humans is disrupted by a millennia-old mutation causing intellectual disability. Mol Cell Biol. PMID: 31263000
• Reichle VF, Kaiser S, Heiss M, Hagelskamp F, Borland K, Kellner S. (2018) Surpassing limits of static RNA modification analysis with dynamic NAIL-MS, Methods, PMID: 30395967

Tasks

Development of synthetic strategies for the synthesis of novel modified ribonucleosides

Synthesis and characertisation of novel modified nucleosides

Synthesis of nucleic acid components for cell culture media

Research interest:

– rRNA removal from total RNA

– fractionation of cellular RNA in the context of NAIL-MS

Topic: New tools for RNA modification dynamics analysis

Elucidation of the mechanisms involved in RNA modifications are next frontiers in molecular biology.
The presence of RNA modifications in many species point towards evolutionarily conserved molecular toolboxes that may modulate the flow of genetic information or allow reacting to environmental challenges. Considerable insight has been gained concerning modification profiles and the fate of modifications by the use of NAIL-MS, which was established by our group.
My goal consists of the investigation of RNA modification profiles and the elucidation of underlying mechanisms of their adaptation during stress response. I am aiming at determining the stress response of yeast to certain chemicals and the fate of RNA modifications, by application of dynamic NAIL-MS, consisting of isotope labeling in pulse-chase experiments followed by LC-MS analysis of nucleosides.

Publications

2022

• ongoing

2021

• Yoluç Y, van de Logt E., Kellner-Kaiser S., The Stress-Dependent Dynamics of S. cerevisiae tRNA and rRNA Modification Profiles. Genes (Basel) PMID: 34573326
• Kaiser S, Byrne SR, Ammann G, Asadi Atoi P, Borland K, Brecheisen R, DeMott MS, Gehrke T, Hagelskamp F, Heiss M, Yoluc Y, Liu L, Zhang Q, Dedon PC, Cao B, Kellner S. Strategies to avoid artifacts in mass spectrometry-based epitranscriptome analyses. Angew Chem Int Ed Engl. PMID: 34339593
• Yoluc, Y. et al. , Instrumental analysis of RNA modifications, Critical Reviews In Biochemistry & Molecular Biology PMID: 33618598
• Heiss, M. et al. Quantification of modified nucleosides in the context of NAIL-MS, Methods in Mol. Bio. PMID: 34085252

Research interest:

– Nucleases for RNA fragment generation

– Chemical reactivity of modified nucleosides