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Published on: May 14, 2020
Perturbation of m6A writers reveals two distinct classes of mRNA methylation at internal and 5' sites
Schraga Schwartz1, Maxwell R Mumbach1, Marko Jovanovic1
1Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
Abstract:
N6-methyladenosine (m6A) is a common modification of mRNA with potential roles in fine-tuning the RNA life cycle. Here, we identify a dense network of proteins interacting with METTL3, a component of the methyltransferase complex, and show that three of them (WTAP, METTL14, and KIAA1429) are required for methylation. Monitoring m6A levels upon WTAP depletion allowed the definition of accurate and near single-nucleotide resolution methylation maps and their classification into WTAP-dependent and -independent sites. WTAP-dependent sites are located at internal positions in transcripts, topologically static across a variety of systems we surveyed, and inversely correlated with mRNA stability, consistent with a role in establishing "basal" degradation rates. WTAP-independent sites form at the first transcribed base as part of the cap structure and are present at thousands of sites, forming a previously unappreciated layer of transcriptome complexity. Our data shed light on the proteomic and transcriptional underpinnings of this RNA modification.
Insights
N6-methyladenosine (m6A) RNA modification is regulated by METTL3 complex proteins. This study maps m6A sites, revealing distinct WTAP-dependent and -independent patterns influencing mRNA stability and transcriptome complexity.
Area of Science:
- Molecular Biology
- Epigenetics
- Transcriptomics
Background:
- N6-methyladenosine (m6A) is a prevalent mRNA modification.
- m6A plays a role in regulating the RNA life cycle.
- The protein complex responsible for m6A methylation is not fully characterized.
Purpose of the Study:
- To identify proteins interacting with METTL3, a key component of the m6A methyltransferase complex.
- To investigate the roles of these interacting proteins in m6A methylation.
- To map m6A methylation sites and classify them based on regulatory dependencies.
Main Methods:
- Protein-protein interaction studies to identify the METTL3 interactome.
- Depletion of specific proteins (WTAP, METTL14, KIAA1429) to assess their requirement for m6A methylation.
- High-resolution mapping of m6A levels to generate methylation maps.
- Analysis of m6A site location, stability, and correlation with transcript features.
Main Results:
- A dense network of proteins interacting with METTL3 was identified.
- WTAP, METTL14, and KIAA1429 were confirmed as essential for m6A methylation.
- Accurate, near single-nucleotide resolution m6A methylation maps were generated.
- Two distinct classes of m6A sites were defined: WTAP-dependent (internal, stable, inversely correlated with mRNA stability) and WTAP-independent (at the first transcribed base, part of the cap structure).
- WTAP-independent sites represent a significant layer of transcriptome complexity.
Conclusions:
- The study elucidates the proteomic and transcriptional regulation of m6A RNA modification.
- WTAP-dependent m6A sites contribute to basal mRNA degradation rates.
- WTAP-independent m6A sites reveal a novel aspect of transcriptome complexity.
- This work provides a framework for understanding the functional landscape of m6A modifications.
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