Mass spectrometry of novel DNA & RNA modifications

2 PhD projects offered in the IPP winter call 2020/2021

 

 

PhD project 1: RNA Epitranscriptomics & Embryonic Stem Cells

Some 170 RNA modifications have been described on different types of RNA, most of which remain poorly understood. Some RNA modifications are present on messenger RNA where they can play a critical role in post-transcriptional gene regulation. Similar to epigenetic marks on the DNA and Histones, these modifications are often highly conserved and can be dynamically regulated in development and disease. This new layer of gene expression regulation has been coined as “epitranscriptomics”.

Identifying novel effectors of the mammalian RNA methylome would result in new insights into transcription, translation, splicing, and various other RNA-mediated cellular processes. Using liquid chromatography-coupled mass spectrometry (LC-MS/MS), we devised a method to screen for effectors of ~50 RNA modifications. Our screens identified several hits for modifying enzymes and the corresponding RNA modifications. 

In this project, you will characterize these RNA modifying enzymes and the RNA modifications. You will monitor the modifications by LC-MS/MS. You will study their role in embryonic stem cell self-renewal and pluripotency, as well as in differentiation. You will create mutants using CRISPR/Cas9 mediated genome editing and characterize them. You will use Next generation sequencing methods and carry out bioinformatics analyses to learn about affected genes and regulatory networks.

PhD project 2: Mass Spectrometry of Epigenetic DNA Modifications

Not only RNA but also DNA is modified in cells to regulate gene activity epigenetically. For example 5’-methylcytosine (5mC) serves as an epigenetic modification, which provides a memory state for silencing gene expression. We utilize highly sensitive mass spectrometry (Triple Quadrupole Mass Spec) and non-radioactive stable isotope metabolic labelling to profile DNA modifications, including 5mC, 5hmC, 5fC, 5caC, m6A, and others.

The successful candidate will have a strong background in Organic Chemistry or Biochemistry to study nucleic acid modifying enzymes to unravel how chemical modifications in genomic DNA and DNA:RNA hybrids (“R-loops”) regulate pluripotency and differentiation of mammalian embryonic stem cells and embryos. You will use LC-MS/MS, genome-edited CRISPR/Cas9 stem cell mutants, as well as various NGS protocols to carry out a multi-dimensional analysis to address: Which modifications can be identified and how do they change with different cellular states? What enzymes mediate them? Where in the genome do the modifications occur? What are molecular determinants involved in targeting of modifications to specific sites? Critical methods you will use are LC-MS/MS, embryonic stem cell self-renewal and pluripotency, CRISPR/Cas9 mediated genome editing, as well as Next generation sequencing (NGS) methods. The project will involve strong interaction with computational biologists.

Publications relevant to the projects

Musheev MU, Baumgärtner A, Krebs L, Niehrs C. (2020). The origin of genomic N6-methyl-deoxyadenosine in mammalian cells. Nat. Chem Biol. doi: 10.1038/s41589-020-0504-2.

Leismann, J., et al (2020) The 18S ribosomal RNA m6A methyltransferase Mettl5 is required for normal walking behavior in Drosophila. EMBO Rep. 21(7):e49443

Contact

Christof Niehrs

Email

Webpage: www.imb-mainz.de/niehrs