m6A regulates breast cancer proliferation and migration through stage-dependent changes in Epithelial to Mesenchymal Transition gene expression.

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.

Assessing 2'-O-Methylation of mRNA Using Quantitative PCR.

2'-O-methylation (Nm) is an RNA modification commonly found on rRNA and snRNA, and at the mRNA 5'-cap, but has more recently been found internally on mRNA. The study of internal Nm modifications on mRNA is in the early stages, but we have reported that this sort of Nm modification can regulate mRNA abundance and translation. Although there are many methods to determine the presence of Nm on rRNA, detecting Nm on specific mRNA transcripts is technically difficult because they are much less abundant than rRNA. Some of these methods rely on the fact that Nm modification of RNA disrupts reverse transcription reactions when performed at low dNTP concentrations. In this chapter, we describe our approach to using quantitative PCR in conjunction with reverse transcription at low dNTPs, which is sensitive enough to detect changes to Nm modification of mRNA.

Modification of messenger RNA by 2'-O-methylation regulates gene expression in vivo.

Epitranscriptomic modifications of mRNA are important regulators of gene expression. While internal 2'-O-methylation (Nm) has been discovered on mRNA, questions remain about its origin and function in cells and organisms. Here, we show that internal Nm modification can be guided by small nucleolar RNAs (snoRNAs), and that these Nm sites can regulate mRNA and protein expression. Specifically, two box C/D snoRNAs (SNORDs) and the 2'-O-methyltransferase fibrillarin lead to Nm modification in the protein-coding region of peroxidasin (Pxdn). The presence of Nm modification increases Pxdn mRNA expression but inhibits its translation, regulating PXDN protein expression and enzyme activity both in vitro and in vivo. Our findings support a model in which snoRNA-guided Nm modifications of mRNA can regulate physiologic gene expression by altering mRNA levels and tuning protein translation.