Perturbation of m6A writers reveals two distinct classes of mRNA methylation at internal and 5' sites

Cell Rep. 2014 Jul 10;8(1):284-96. doi: 10.1016/j.celrep.2014.05.048.

Schraga Schwartz ¹, Maxwell R Mumbach ¹, Marko Jovanovic ¹, Tim Wang ², Karolina Maciag ³, G Guy Bushkin ⁴, Philipp Mertins ¹, Dmitry Ter-Ovanesyan ¹, Naomi Habib ¹, Davide Cacchiarelli ⁵, Neville E Sanjana ¹, Elizaveta Freinkman ⁴, Michael E Pacold ⁶, Rahul Satija ¹, Tarjei S Mikkelsen ⁵, Nir Hacohen ⁷, Feng Zhang ⁸, Steven A Carr ¹, Eric S Lander ⁹, Aviv Regev ¹⁰

Affiliations

1 Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
2 Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
3 Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Graduate Program in Immunology, Division of Medical Sciences, Harvard Medical School, Boston, MA 02115, USA.
4 Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA.
5 Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Harvard Stem Cell Institute and Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA.
6 Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA; Department of Radiation Oncology, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, MA 02215, USA.
7 Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Center for Immunology and Inflammatory Diseases, Massachusetts General Hospital, Charlestown, MA 02129, USA.
8 Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Brain and Cognitive Sciences and Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
9 Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Systems Biology, Harvard Medical School, Boston, MA 02114, USA. Electronic address: lander@broadinstitute.org.
10 Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Howard Hughes Medical Institute, 4000 Jones Bridge Road, Chevy Chase, MD 20815, USA. Electronic address: aregev@broad.mit.edu.

PMID: 24981863 DOI: 10.1016/j.celrep.2014.05.048

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.

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