Andrea Rentmeister’s team detects methylated sites with click chemistryThe genetic information of an organism is stored in the form of DNA (deoxyribonucleic acid) in every cell. In order to be able to produce proteins with this blueprint, the DNA is first transcribed into so-called mRNA (messenger ribonucleic acid). Like DNA, this consists of four basic building blocks. These basic building blocks can be naturally chemically modified at certain points and thus influence cell development and cell metabolism. For example, such modifications change the stability of RNA and control the specialization of cells for certain tasks, the quantity of proteins produced or the growth of tumours. A team led by biochemist Andrea Rentmeister from the University of Münster and bioinformatician Christoph Dieterich from Heidelberg University Hospital presents a new method in the journal "Nature Communications" for comprehensively detecting and precisely localizing these modifications in mRNA.
In contrast to mutations, the modifications do not change the genes themselves. "However, if modifications are incorrectly incorporated into the basic building blocks of the mRNA, this can have dramatic consequences for the cell," emphasizes first author Nadine Kück, a doctoral student in Andrea Rentmeister’s research group. "It is therefore important to understand what effects the individual modifications have. We need to know exactly where the modifications are located on a gene, how frequently they occur there and how healthy and diseased cells differ in this respect. Perhaps one day it will be possible to treat diseases caused by faulty modifications."
The most important type of modification is the labeling of molecules of the genetic material with so-called methyl groups. This methylation can occur on all basic building blocks of DNA and mRNA as well as on different sites of these basic building blocks. In the mRNA of animals, plants and fungi - so-called eukaryotes - the 6-methyladenosine and the 5-methylcytosine are particularly important within the methylations. With the new method now presented, these sites can be determined in a single experiment for all RNA molecules produced in the cell and precisely located on the respective gene. The trick: instead of the antibodies that are usually used, the researchers use a metabolic label in which a molecular recognition mark is incorporated into the mRNA - in this case propargyl groups instead of methyl groups.
The advantage is that the propargyl groups are accessible via click chemistry. "This means that they are starting points for fast and precise reactions. However, as the required reaction partners are not present in the cell, they do not react within the cell, which is why they are biocompatible for the cell," says Nadine Kück. "In this way, we can mark them in such a way that we can easily separate the mRNA with the propargyl groups from the other cell components and ultimately identify the exact sites with the modifications on the genes."
Antibodies are often used in other studies to label modified areas of RNA. However, scientists are trying to find alternatives, as labeling with antibodies is not always very accurate and can lead to false positive and false negative results.
The work was funded by the priority program SPP1784 (RE 2796/3-2, DI 1501/11-1) of the German Research Foundation (DFG) and by a scholarship of the graduate program CiM-IMPRS.
Hartstock, K., Kueck, N.A., Spacek, P. et al. (2023): MePMe-seq: antibody-free simultaneous m6A and m5C mapping in mRNA by metabolic propargyl labeling and sequencing. Nat Commun 14, 7154. DOI:10.1038/s41467’023 -42832-z