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METTL16, a human m6A RNA methyltransferase, is currently known for its modification of U6 and MAT2A RNAs. Several studies have identified additional RNAs to which METTL16 binds, however whether METTL16 modifies these RNAs is still in question. Moreover, a recent study determined that METTL16 contains more than one RNA-binding domain, leaving the importance of each individual RNA-binding domain unknown. Here we examined the effects of mutating the METTL16 protein in certain domains on overall cell processes. We chose to mutate the N-terminal RNA-binding domain, the methyltransferase domain, and the C-terminal RNA-binding domain. With these mutants, we identified changes in RNA-binding ability, protein and RNA expression, cell cycle phase occupancy, and proliferation. From the resulting changes in RNA and protein expression, we saw effects on cell cycle, metabolism, intracellular transport, and RNA processing pathways, which varied between the METTL16 mutant lines. We also saw significant effects on the G1 and S phase occupancy times and proliferative ability with some but not all the mutants. We have therefore concluded that while METTL16 may or may not m6A-modify all RNAs it binds, its binding (or lack of) has a significant outcome on a variety of cell processes.

Methyltransferases are a wide-ranging, yet well-conserved, class of molecules that have been found to modify a wide variety of substrates. Interest in RNA methylation has surged in recent years with the identification of the major eukaryotic mRNA m6A methyltransferase METTL3. METTL16 has also been identified as an RNA m6A methyltransferase; however, much less is known about its targets and actions. Interestingly, in addition to their catalytic activities, both METTL3 and METTL16 also have “methylation-independent” functions, including translational regulation, which have been discovered. However, evidence suggests that METTL16’s role as an RNA-binding protein may be more significant than is currently recognized. In this review, we will introduce RNA methylation, specifically m6A, and the enzymes responsible for its deposition. We will discuss the varying roles that these enzymes perform and delve deeper into their RNA targets and possible roles as methylation-independent RNA binding proteins. Finally, we will touch upon the many open questions still remaining.

While many factors have been implicated in breast cancer progression, effective treatments are still lacking. In recent years, it has become clear that posttranscriptional regulation plays a key role in the aberrant gene expression underlying malignancy and metastasis. For example, the mRNA modification N6-methyladenosine (m6A) is involved in numerous post-transcriptional regulation processes and has been implicated in many cancer types, including breast cancer. Despite intense study, even within a single type of cancer, there is little consensus, and often conflicting results, as to the role of m6A, suggesting other factors must influence the process. The goal of this study was to determine if the effects of m6A manipulation on proliferation and migration differed based on the stage of disease progression. Using the MCF10 model of breast cancer, we reduced m6A levels by targeting METTL3, the main cellular m6A RNA methyltransferase. Knocking down Mettl3 at different stages of breast cancer progression indeed shows unique effects at each stage. The early-stage breast cancer line showed a more proliferative phenotype with the knockdown of Mettl3 while the transformed breast cancer line showed a more migratory phenotype. Interestingly, the metastasized breast cancer cell line showed almost no effect on phenotype with the knockdown of Mettl3. Furthermore, transcriptome wide analysis revealed EMT as the probable pathway influencing the phenotypic changes. The results of this study may begin to address the controversy of m6A’s role in cancer and suggest that m6A may have a dynamic role in cancer that depends on the stage of progression.