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

PROJECT SUMMARY
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.

Grant Number: 1R15GM152920-01
NIH Institute/Center: NIH
Principal Investigator: Sanjaya Abeysirigunawardena