The existence of RNA nucleotide modifications in functional RNAs is 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 shown to impact viral infections, sperm maturation, and cancer progression. In cells, m6A levels are controlled by methyl writers and readers. These proteins codes the stress signal on to mRNA transcripts, both post-, and co-transcriptionally. Methyl readers that recognize methylations play a critical role in decoding stress signals and direct mRNA to either getting edited, processed, degraded, or translated. Given the broader diversity of mRNA methylation states under various stress conditions and in human diseases, an assemblage of methyl readers that can read 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 up 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. In this research, the phage display method is used to discover a sequence or structural consensus essential for m6A recognition. The impact of RNA structure and sequence on the sequence or structure consensus will also be tested. Sequence comparison will be made between the peptides enriched against methylated RNA targets (phage display) and proteins identified from pulldown assays. Preliminary result of this research illustrates that 1) RNA methylations enhance sequence-specific interactions of RNA with proteins, 2) two tryptophan residues that reside four amino acid residues apart in the peptide may play a role in m6A recognition, and 3) RNA binding sites of writer or eraser proteins have similar sequences as the selected peptides against unmodified and modified RNA targets, respectively. Long term objectives of this project include engineering unique designer proteins in which m6A-recognizing peptides (that binding sequence specifically or structure specifically) are fused with proteins related to RNA processing, localization, and degradations to use in treating human diseases.
Contrasting levels of N6-methyladenosine (m6A) in cellular mRNAs are observed in various human diseases such as cancer. Our goal is to study the ability of m6A to change RNA-protein interactions and RNA folding that are critical for epitranscriptomic regulation of gene expression using various biophysical and biochemical methods, including phage display. With the successful completion of this project, we will discover novel RNA-protein interactions that are important for epitranscriptomic regulation of gene expression.
