Full western blot pictures.

<div><p>RNA-modifying enzymes have recently garnered considerable attention due to their relevance in cancer biology, identifying them as potential targets for novel therapeutic intervention. THUMPD3 was recently identified as an RNA methyltransferase catalysing N<sup>2</sup>-methylguanosine (m<sup>2</sup>G) within certain tRNAs. In this study, we unveil a novel role for THUMPD3 in lung cancer cells. Depletion of the enzyme from lung cancer cells significantly impairs their fitness, negatively impacting key cellular processes such as proliferation and migration. Notably, exogenous expression of THUMPD3 in normal lung fibroblasts stimulates their proliferation rate. Additionally, transcriptome-wide analyses reveal that depletion of THUMPD3 from lung cancer cells induces substantial changes in the expression of cell surface proteins, including those comprising the extracellular matrix (ECM). We further demonstrate that THUMPD3 maintains expression of an extra-domain B (EDB) containing pro-tumour isoform of <i>Fibronectin-1</i> mRNA, encoding FN1, an important ECM protein. Crucially, depletion of THUMPD3 promotes an alternative splicing event that removes the EDB-encoding exon from <i>Fibronectin-1</i>. This is consistent with THUMPD3 depletion reducing cellular proliferation and migration. Moreover, depletion of THUMPD3 selectively and preferentially affects the alternative splicing of ECM and cell adhesion molecule encoding transcripts, as well as those encoding neurodevelopmental proteins. Overall, these findings highlight THUMPD3 as an important player in regulating cancer-relevant alternative splicing and they provide a rationale for further investigations into THUMPD3 as a candidate target in anti-cancer therapy.</p></div>