Investigation of the key structural and sequence features essential for m6A recognition during post-transcriphonal gene expression

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PROJECT SUMMARY The presence of RNA nucleotide modifications in functional RNAs has been known for many decades. Several recent studies illustrate the transcriptome-wide presence of nucleotide modifications such as pseudouridines, N6-methyladenosines (m6A), and 5-methylcytosines. The levels of nucleotide modifications in mRNA are in tight equilibrium unless cells are under various stress conditions. Changes in m6A levels in mRNA have impacted viral infections, sperm maturation, and cancer progression. In cells, m6A levels are controlled by methyl writers and readers. These proteins codes the stress signal onto mRNA transcripts, both post- and co-transcriptionally. Methyl readers that recognize methylations play the critical role of decoding stress signals and either edit, process, degrade or translate mRNA. Given the broader diversity of mRNA methylation states under various stress conditions and in human diseases, an assemblage of methyl readers capable of reading each unique stress signal should exist. The lack of general structural and sequence consensus for methyl-recognizing proteins (reader or erasers) impedes the discovery of novel regulation mechanisms by readers and erasers not known to date. The three short term goals of this project are 1) to discover sequence or structural consensus for short peptides that interact with m6A, 2) to understand how RNA structure and sequence can change the sequence and the structure of m6A-recognizing peptides, 3) to investigate the ability of enriched peptides to inhibit reader and eraser protein. We used the phage display method to discover a general sequence or structural consensus for proteins recognizing nucleotide methylations. We propose to test the impact of RNA structure and sequence on the sequence or structure of the enriched peptides. Our pull-down assays will evaluate the potential of the enriched peptides to mimic known methyl readers. We also propose to compare the peptides selected against methylated targets (phage display) and proteins identified from pull-down assays for sequence similarity. Our preliminary work shows that 1) RNA methylations enhance the RNA sequence-specific interactions with proteins, 2) two tryptophan residues that reside four amino acid residues apart may play a more significant role in m6A recognition 3) RNA binding sites of writer or eraser proteins have similar sequences as the selected peptides against unmodified and modified RNA targets, respectively. Isothermal calorimetry (ITC) is a powerful technique to study the affinity of biomolecules to each other and various small molecular ligands. We propose using ITC to measure peptides' binding affinity to various modified and unmodified RNA oligonucleotides. These experiments will allow us to quantify peptides' binding specificity or preference to methylated RNA oligonucleotides. In addition, we will use ITC to validate protein enriched in our RNA pull-down assays. This research will help identify fundamental sequence properties of proteins/peptides that enable recognizing m6A methylations in RNA.