Abstract
N6-methyladenosine (m6A) is the most abundant modification in mRNA. The core of the human N6-methyltransferase complex (MTC) is formed by a heterodimer consisting of METTL3 and METTL14, which specifically catalyzes m6A formation within an RRACH sequence context. Using recombinant proteins in a site-specific methylation assay that allows determination of quantitative methylation yields, our results show that this complex methylates its target RNAs not only sequence but also secondary structure dependent. Furthermore, we demonstrate the role of specific protein domains on both RNA binding and substrate turnover, focusing on postulated RNA binding elements. Our results show that one zinc finger motif within the complex is sufficient to bind RNA, however, both zinc fingers are required for methylation activity. We show that the N-terminal domain of METTL3 alters the secondary structure dependence of methylation yields. Our results demonstrate that a cooperative effect of all RNA-binding elements in the METTL3–METTL14 complex is required for efficient catalysis, and that binding of further proteins affecting the NTD of METTL3 may regulate substrate specificity.
Funding source: Deutsche Forschungsgemeinschaft
Award Identifier / Grant number: SFB902
Acknowledgments
This work was funded by DFG in SFB902 “Molecular Principles of RNA-based Regulation”, and by Goethe University Frankfurt. The authors would like to thank Prof. Harald Schwalbe for constant support. Gerd Hanspach is acknowledged for providing the OT RNA, Dr. Santosh Gande for help with protein expression, and Kaili Wang for assistance in CD-spectroscopy measurements.
Author contribution: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.
Research funding: This work was funded by Deutsche Forschungsgemeinschaft (SFB902).
Conflict of interest statement: The authors declare no conflicts of interest regarding this article.
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Supplementary material
The online version of this article offers supplementary material (https://doi.org/10.1515/hsz-2020-0265).
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