SRP410867 PRJNA907315 GSE219172 The post-translational modifications and translation-regulatory role of METTL14 in 5-fluorouracil-resistant colorectal cancer [Ribo-seq] 5-fluorouracil (5-FU) based chemotherapy is the first-line chemotherapy scheme for CRC, whereas acquired drug resistance poses a huge obstacle to curing CRC patients and the mechanism is still obscure. METTL14 is a core subunit of the m6A methyltransferase complex and has been reported to regulate the drug resistance of cancers. Here, we demonstrated that METTL14 is significantly downregulated in 5-FU-resistant CRC and METTL14 overexpression inhibits the proliferation and 5-FU resistance of CRC cells in vitro and in vivo. In addition, we found that MIB1 catalyzes the K63-linked ubiquitination of METTL14 and induces its degradation through the autolysosome pathway, while AURKA-mediated phosphorylation enhances METTL14 stability by inhibiting its ubiquitination. Moreover, we first discovered that METTL14 functions in 5-FU-resistant CRC cells by directly participating in the translation initiation of non-m6A-modified mRNAs independently of METTL3, rather than cooperating with METTL3 to methylate adenosine residues of targets. Further analysis indicated that METTL14 regulates the cell cycle process in 5-FU-resistant CRC cells. Collectively, our study not only identified METTL14 as a potential therapeutic target for 5-FU resistance in CRC but also revealed a novel translation-facilitating role of METTL14. Overall design: METTL14 overexpression plasmids and negative control plasmids were transfected into 5-FU-resistant HCT-8 cells, respectively. Then cells were treated with 100ug/mL CHX and implemented to the Ribosome-sequencing. Experiments were performed twice independently. GSE219172 parent_bioproject PRJNA907307