RNA-Binding Peptide Influences Epitranscriptomic Regulation by Preferentially Binding to Unmodified RNAs Targeted by...

5-Methylcytosine (m⁵C) is a widespread mRNA modification that regulates gene expression and is frequently dysregulated in cancer. Here, we used phage display to identify peptides that bind an NSUN2 consensus RNA sequence in either its unmodified (UN2-RNA) or m⁵C-modified (MN2-RNA) form. A single peptide, un2p1 (TDYSTHRLSHSL), was selectively enriched against both targets. Biophysical analyses demonstrated that un2p1 binds this RNA sequence with high affinity and that m⁵C incorporation reduces binding by approximately ninefold, indicating that cytidine methylation acts as a biochemical switch for peptide-RNA recognition. Sequence alignments mapped un2p1 to a conserved structural loop and catalytic domain region within NSUN2, suggesting that the peptide functions as a molecular mimic of the endogenous RNA-recognition interface. In A549 lung adenocarcinoma cells, un2p1 treatment reduced global m⁵C levels and downregulated the oncogenic chaperonin CCT5, consistent with disruption of NSUN2-dependent Wnt/β-catenin signaling. These effects were attenuated in non-malignant HEK293 cells, which exhibited a compensatory increase in m⁵C levels and preserved viability. Together, these findings identify un2p1 as a sequence-specific, methylation-sensitive RNA-binding peptide that modulates the m⁵C epitranscriptome and selectively impairs lung cancer cell viability, highlighting a substrate-centric strategy for targeting NSUN2-mediated oncogenic pathways.