The impact of aberrant m5C mRNA modification on translation initiation and cancer

Post-transcriptional modifications on mRNAs are critical for modulating protein synthesis and maintaining cellular homeostasis. mRNAs are decorated with numerous chemical modifications, including 5- methylcytosine (m5C), which is enriched in regulatory 5¢ untranslated regions (5¢-UTRs). Aberrant m5C methylation caused by overexpressed methyltransferases in cancer cells is known to stabilize specific oncogenic mRNAs, thereby promoting cell proliferation and tumorigenesis. However, how aberrant m5C methylation of 5¢-UTRs affects translation initiation and dysregulates protein synthesis remains largely unknown. Dysregulated translation of oncogenes and tumor suppressors is likely to contribute to the pathogenic effects of dysregulated methyltransferase expression in tumors. The primary objective of this proposal is to determine the regulatory role of dose-sensitive m5C modification in the early stages of translation and uncover its direct impact on ribosome recruitment to mRNA 5¢-UTRs. To study physiological m5C residues, I first purified the key mRNA modifying members of the NOL1/NOP2/SUN (NSUN) family of methyltransferases, NSUN2 and NSUN6, and verified their methyltransferase activities using known targets. Here, I will screen ~35,000 human endogenous 5¢-UTR sequences from multiple tissues, encompassing all genetic isoforms, for m5C installation by NSUN2 and NSUN6. My preliminary computational analysis indicates the presence of thousands of potential m5C targets in this 5¢-UTR pool. The overarching hypothesis of this proposal is that m5C is dose-dependently installed on 5¢-UTRs and quantitively impacts translation initiation. To test this hypothesis, I will employ an innovative high-throughput approach developed in our lab, direct analysis of ribosome targeting (DART), to quantify ribosome recruitment on UTR pools methylated with varying concentrations of NSUN2 or NSUN6. Aim 1 will identify dose-sensitive m5C targets through bisulfite sequencing, measure m5C-dependent changes in ribosome recruitment, and validate methylation-sensitive translation activity in cancer cells. Aim 2 will identify and characterize trans-acting RNA binding proteins that modulate m5C-mediated translational control. Together, the proposed work will elucidate how aberrant m5C methylation, caused by overexpression of NSUN2 and NSUN6, mediates dysregulation of translation in cancer cells. My results are expected to reveal novel and fundamental mechanisms of translation initiation in human cells and may uncover new therapeutic targets for cancers with overexpressed NSUN2 and NSUN6. Project Number: 1F32CA306485-01 | Fiscal Year: 2025 | NIH Institute/Center: National Cancer Institute (NCI) | Principal Investigator: HeaJin Hong | Institution: YALE UNIVERSITY, NEW HAVEN, CT | Award Amount: $75,052 | Activity Code: F32 | Study Section: Special Emphasis Panel[ZRG1 F08-L (20)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11241711