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

> **NIH NIH R15** · KENT STATE UNIVERSITY · 2022 · $100,000

## Abstract

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
seque...

## Key facts

- **NIH application ID:** 10582122
- **Project number:** 3R15GM137291-01A1S1
- **Recipient organization:** KENT STATE UNIVERSITY
- **Principal Investigator:** Sanjaya Chinthaka Abeysirigunawardena
- **Activity code:** R15 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $100,000
- **Award type:** 3
- **Project period:** 2020-09-15 → 2023-09-14

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10582122

## Citation

> US National Institutes of Health, RePORTER application 10582122, Investigation of the key structural and sequence features essential for m6A recognition during post-transcriphonal gene expression (3R15GM137291-01A1S1). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10582122. Licensed CC0.

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