Project Details
Projekt
Fine-mapping of mRNA m6A sites in zebrafish development
Subject Area
Biochemistry
Term
from 2015 to 2019
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 277312457
Reversible methylation of mRNA at position N6 of adenosine (m6A) is the most abundant non-cap modification in mRNAs of metazoans. Findings in different organisms have emphasized its importance for multiple processes during germ cell development and embryogenesis. However, the exact role of m6A during development is still poorly understood. To overcome this lack of knowledge, it will be crucial to obtain a detailed and quantitative picture of m6A sites in different cell-types and developmental stages. This requires the precise and direct identification of m6A sites. Although m6A sites can be mapped on the transcriptome-wide level using m6A-specific antibodies in combination with photo-crosslinking, these methods are indirect and require high sequencing depth. Direct quantification of m6A is possible, but not applicable to transcriptome-wide analyses. To overcome these limitations, we developed a novel chemical biology approach for direct and precise mapping of m6A sites. Our approach utilizes synthetic analogs of the cosubstrate S-adenosyl-L-methionine (AdoMet), which are used by METTL3-METT14. We showed that non-natural modifications in combination with click chemistry block reverse transcription. The resulting RNA samples can be used to generate sequencing libraries for the precise assignment of m6A sites.Based on these achievements we are now implementing our approach in mammalian cells and zebrafish to investigate the functions of METTL3-mediated methylation during development. Furthermore, we aim to investigate the methylation dynamics under stress condition in mammalian cells by generating pulses of the non-natural modification. Comparing sequencing libraries from different samples will potentially allow for relative quantification of m6A on a transcriptome-wide level.
DFG Programme
Priority Programmes
International Connection
Switzerland
