Several post-transcriptional modifications are present in eukaryotic mRNA. N6-methyladenosine (m6A) is the most common modification throughout the mammalian RNA transcriptome and is highly prevalent in mRNAs, as well as in certain non-coding RNAs. It is found most often near stop codons and in 3’ untranslated regions of mRNA, but is also found within mRNA exons expressing the consensus sequence, RRACH, where R = purine, A = m6A, and H = A, C, or U. The m6A modification regulates different stages of mRNA metabolism, including folding, maturation, export, translation, and decay. This, in turn, drives numerous biological processes, including circadian rhythms, T-cell differentiation, embryonic stem cell renewal and differentiation, epithelial-mesenchymal transition, adipogenesis, and cortical neurogenesis.
The addition of m6A to mRNA is a reversible modification, regulated by a cyclic enzymatic reaction catalyzed by writers and erasers. Writers, or methyltransferases, install the modifications, and erasers, or demethylases, remove them. Examples of writers include the complex, METTL3/METTL14, as well as METTL16. Examples of demethylases, include fat mass and obesity-associated (FTO) protein, an obesity susceptibility factor that is also an RNA N6-methyladenine demethylase, and AlkB homolog 5 (ALKBH5). The METTL3/METTL14 is a nuclear methyltransferase complex composed of multiple subunits, including METTL3, METTL14, and Wilms tumor 1-associating protein (WTAP). METTL3 is the methyltransferase within this complex. METTL14 acts as an adaptor, binding the substrate and promoting methyltransferase activity. WTAP directs the complex to mRNA targets in the nucleus and supports catalytic activity.
Readers are proteins exerting regulatory effects on mRNA metabolism by selectively binding to m6A. There are several families of m6A-binding proteins. One such family is the YTH domain-containing proteins, which can be divided into three major classes: YTHDC1, YTHDC2, and YTHF proteins. YTHDC1 proteins are found in the nucleus, directing mRNA splicing, whereas YTHDC2 and YTHDF proteins are predominantly cytoplasmic, mediating translational efficiency and decay of m6A-modified mRNAs. Three paralogs (DF1, DF2, and DF3) comprise YTHDF proteins. The eIF3 family of proteins assembles on the 40S subunit and promotes cap-independent translation, either by direct binding to m6A within 5’ UTRs of mRNAs or through a yet to be defined mechanism involving YTHDF1.
Other readers include HNRNPA2B1, HNRNPC, HNRNPG, and insulin-like growth factor-2 mRNA binding proteins 1, 2, and 3 (IGF2BP1/2/3), all found in the nucleus. For these RNA-binding proteins, the m6A modification causes structural switching in the mRNA, which allows binding of HNRNPC and HNRNPG; or IGF2 binding proteins to m6A-adjacent sites to mediate mRNA stability and export, respectively. Furthermore, HNRNPA2B1 also binds through a switch mechanism to regulate primary miRNA maturation, either by direct binding to m6A or by binding to a nearby non-methylated consensus sequence.
Several studies suggest that changes in m6A modification patterns are implicated in tumorigenesis, leading to numerous cancers, including breast cancer, lung cancer, acute myeloid leukemia, glioblastoma, and more. For example, in breast cancer stem cells, ZNF217 reportedly interacts with METTL3. This, in turn, may inhibit the m6A of two gene transcripts, KLF4 and NANOG, increasing their expression and promoting tumor progression. Additionally, in lung cells, SUMO1 catalyzes the post-translational modification of METTL3, thereby decreasing m6A modification and promoting the development of non-small-cell lung carcinoma. Furthermore, elevated FTO levels have been reported in hematopoietic stem cells, downregulating m6A of mRNA transcripts involved in hematopoietic cell transformation, ASB2 and RARA. These are just some of the many examples of how deregulation of m6A modification may influence tumorigenesis. Further study of this pathway may pave the way to novel and effective tumor therapies.
We would like to thank Dr. Samie R. Jaffrey, Department of Pharmacology, Weill Medical College, Cornell University, for reviewing this pathway.