Project Summary
The goal of this project is to determine how phosphorylation of METTL3 by ERK2 regulates N6-
methyladenosine (m6A) methyltransferase activity to affect the tumorigenicity of melanoma cells. Post-
transcriptional RNA modifications are a major component of regulating gene expression. N6-methyladenosine
(m6A) is the most abundant internal mRNA and long non-coding RNA modification. Through its effects on RNA
stability, structure, export, splicing, and translation, m6A influences many biological processes, including
embryonic development, immune response, memory, viral infection, and cancer. m6A is selectively installed on
certain mRNA transcripts by the methyltransferase-like 3 (METTL3), which acts as the catalytic subunit of the
mammalian m6A methyltransferase “writer” complex, and must be precisely regulated for proper biological
function. For example, endometrial cancer often contains reduced METTL3 expression, which leads to reduced
m6A methylation. Reduced m6A methylation then affects mRNA metabolism of certain transcripts, which
ultimately results in increased activation of the AKT pathway and tumorigenicity. Similar METTL3-dependent
phenomena are also observed in leukemia, lung cancer, and embryonic stem cells. While METTL3 can
regulate activation of signaling pathways, it is poorly understood how METTL3 is post-translationally regulated
and how such regulation affects the methyltransferase activity of METTL3. Preliminary results indicate that
activation of ERK2 phosphorylates METTL3, and that lack of METTL3 phosphorylation at these phospho-sites
reduces m6A methylation. In murine embryonic stem cells, METTL3 phosphorylation appears to impact
pluripotency factor expression. Because dysregulation of kinase signaling, including the ERK pathway, often
leads to uncontrolled growth in cancer cells and plays a significant role in tumorigenicity, I aim to determine
how ERK2 phosphorylation of METTL3 affects m6A methyltransferase function. I will use melanoma cells as a
model because they frequently a contain BRAF V600E mutation that constitutively activates the ERK pathway.
With a combination of molecular biology, biochemistry, and genomics, I will accomplish the following aims.
(I) First, I will investigate the effects of METTL3 phosphorylation on the m6A methyltransferase complex
activity. Specifically, I will study changes in enzymatic activity, m6A “writer” complex association, and
subcellular localization. (II) Second, I will determine how METTL3 phosphorylation affects melanoma
malignancy. Because m6A-seq data suggests the apoptotic signaling pathway is differentially methylated due
to METTL3 phosphorylation, I will validate whether the apoptotic pathway is differentially active. Then I will
determine which genes are differentially expressed, and which reader proteins effect those changes in
expression. This work will elucidate a novel layer of post-transcriptional regulation in cancer. A better
understanding of how RNA methylation is dysregulated by kinase signaling in cancers will be valuable for
development of future cancer therapies.