The roles of METTL3 in colorectal cancer

Colorectal cancer (CRC) is a leading cause of cancer-related mortality worldwide, accounting for 10% of all cancer deaths. Metastatic disease occurs in half of all CRC cases, with a 5-year survival rate of 14%. Thus, understanding molecular mechanisms of CRC progression is important for improving diagnostics and therapies. Recent studies identified the m6A (N6-methyladenosine) RNA methyltransferase-METTL3 as a key oncogene in CRC, suggesting the important role of epitranscriptomic regulation in cancer. M6A modification affects multiple aspects of RNA metabolism, including degradation, export, and translation. However, the precise mechanisms of METTL3 in CRC remain undefined. We identified YTHDF1, an m6A reader, as the most upregulated reader in CRC patients. Yet, the METTL3-YTHDF1 interplay in CRC progression is unclear. This study aims to define how METTL3 and YTHDF1 act together to promote CRC progression via m6A modifications, and to identify novel, clinically relevant m6A targets. To achieve this, we performed Nanopore direct RNA-seq in METTL3-knockdown cells to map METTL3-dependent m6A sites across the CRC transcripts. To identify genes whose m6As are read by YTHDF1, we conducted short-read RNA-seq in METTL3- and YTHDF1-knockdown cells to determine shared transcriptional changes associated with RNA methylation. Integrating these datasets with patient data revealed clinically relevant transcripts regulated by METTL3 and YTHDF1. m6A enrichment on selected candidates were validated by MeRIP-qPCR. Comparison with two existing m6A databases confirmed overlapping sites and uncovered previously unannotated sites. Among these, TRIB3 , a known oncogene in CRC, consistently emerged as a top m6A target across datasets, with expression significantly reduced upon METTL3 or YTHDF1 knockdown. Thus, TRIB3 was selected as a conceptual target for further studies. Using RNA immunoprecipitation, we found YTHDF1 interacts with TRIB3 , and this interaction is reduced upon METTL3 depletion. Knockdown of METTL3 or YTHDF1 reduces TRIB3 RNA stability and polysome occupancy causing decreased transcript and protein levels. To directly remove m6As on TRIB3 , we used dCasRX conjugated to ALKBH5, an m6A demethylase, and coupled with sgRNAs targeting TRIB3 . Dual sgRNAs effectively removed m6As on TRIB3 and reduced its expression, confirming functional roles of m6As in regulating TRIB3 level. TRIB3 overexpression rescued migration and invasion defects in METTL3 knockdown cells, while m6A removal on TRIB3 by dCasRX-ALKBH5 suppressed these phenotypes, indicating METTL3 and YTHDF1 promote CRC invasion and migration through TRIB3 regulation. Our study highlights the role of the YTHDF1-TRIB3 axis in cooperating with METTL3 to promote CRC. Additionally, our unbiased analyses reveal several other potential m6A targets in CRC. These findings provide new insights into CRC progression and highlight potential therapeutic paths targeting the m6A regulatory network.