Role of N6-methyladenosine in regulating RNA-protein interactions and its effect on cancer cell viability

Post-transcriptional regulation involving RNA modifications is known as epitranscriptomic regulation. The ability of nucleotide modifications to modulate RNA-protein interaction is the basis of epitranscriptomic regulation. There are over 170 nucleotide modifications in RNA, among them m6A modification is the most abundant nucleotide modification in eukaryotic mRNA. The adenine is methylated by a set of proteins known as writers (METTL3- METTL14 complex) and demethylated by another set of proteins known as erasers (FTO and Alkbh5). Changes in m6A levels due to abnormal expression levels of writers and erasers are associated with various diseases and conditions including cancer. Oncogenicity in cancers with decreased m6A levels due to over-expression of FTO can be reduced by inhibiting FTO. In Chapter 1 of my dissertation work, the role played by m6A modification on cancer cell viability was investigated. In this study, phage display was used to discover a peptide inhibitor that binds with m6A-modified RNA leading to the inhibition of FTO binding to its target RNA. The peptide binds to methylated RNA with high affinity and inhibits FTO binding to methylated target RNA. In addition, m6A levels increased, and cell viability decreased in cancer cells upon treatment with the peptide.The m6A modification resides within a consensus sequence known as DRACH (D=A, G, U; R=A, G; H=A, C, U). The selection pressure exerted by m6A modification and its surrounding nucleotides is not well understood. In chapter 2 of my dissertation work, the evolutionary pressure exerted by m6A modification and its surrounding nucleotides was studied. Although phage display is extensively used as a method to identify novel drugs against protein or RNA targets, in this study intrinsic strength of phage display as a directed evolution method was used to understand the selection pressure exerted by m6A modification and its surrounding nucleotides on its binding proteins. Phage display experiments were conducted using RNA constructs with the most and least abundant DRACH sequences as well as with structured RNA with m6A modification in various secondary structures. These phage display experiments illustrated that m6A-modified RNA exerts a unique selection pressure compared with an unmodified RNA regardless of its DRACH sequence and that m6A modifications can exert different selection pressures depending on the region of the secondary structure of the RNA with m6A modification. There can be many m6A modifications in mRNA and they are dynamic. This dynamic nature is due to the addition or removal of methylations in mRNA by writers and erasers as a response to various intracellular or extracellular signals. This dynamicity in m6A modifications elicits the presence of other m6A readers apart from those currently found in the literature. In the third chapter of my dissertation work, the presence of novel m6A readers that are not reported in the current literature was investigated. To identify novel m6A readers, RNA pull-down assays were conducted using RNA targets that were used in phage display with and without m6A modification. Bioinformatics methods such as multiple sequence alignment and structural analysis were used to identify potential m6A binding proteins. This study identified hnRNP A1 as a potential m6A reader. Dot blot assay and calorimetric titrations illustrated that HNRNPA1 has a higher binding affinity towards the methylated RNA than the unmethylated RNA further proving the ability of hnRNP A1 to act as an m6A reader.