Investigation of the essential structural and sequence features for the recognition of RNA methylations during post-transcriptional regulation of gene expression

This Project Grant award from the National Institute of General Medical Sciences (NIGMS) under CFDA 93.859 Biomedical Research and Research Training provides $454,805 to Kent State University to investigate the structural and sequence features required for recognizing RNA methylations and their role in post-transcriptional regulation of gene expression. The 3-year project aims to: 1) discover consensus sequences or structures for peptides that interact with N6-methyladenosine, 2) understand how RNA structure and sequence impact the recognition by these peptides, and 3) evaluate the ability of the discovered peptides to inhibit known RNA methylation reader and eraser proteins. This research seeks to enhance the understanding of RNA methylation-based regulation mechanisms, with the long-term goal of engineering designer proteins to target RNA processing, localization, and degradation for treating human diseases.

Abstract

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 been shown to impact viral infections, sperm maturation, and cancer progression. In cells, M6A levels are controlled by methyl writers and readers. These proteins code the stress signal on to mrna transcripts, both post-, and CO-transcriptionally. Methyl readers that recognize methylations play the critical role of 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 are 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 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. We use phage display method to discover a general sequence or structural consensus for proteins that recognize nucleotide methylations. We propose to test the impact of RNA structure and sequence on the sequence or structure of the enriched peptides. Our pulldown 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 pulldown 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 greater 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. Our long-term objective is to engineer 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.