PROJECT SUMMARY/ABSTRACT Misregulation of nucleoporins (NUPs), the essential constituents of nuclear pore complexes (NPCs), and dysfunction of N6-methyladenosine (m6A) signaling have been individually linked to cancer. Although it has been reported that NPC-mediated macromolecular transport cooperates with m6A modification to control movement of messenger RNAs (mRNAs) out of the nucleus, contribution of such cooperation to pathogenesis and progression of cancer remains obscure. Our proteomic study of NPC-associated cofactors revealed a direct interaction between the m6A methyltransferase METTL3 and one of the NUP proteins called NUP93 in several cancer cell lines including prostate cancer (PCa). Although each protein has been indicated to involve in the progression of some types of human malignancies, their functions and molecular mechanisms of action are poorly understood in PCa, let alone their interaction. Our immunohistochemistry analysis showed that both METTL3 and NUP93 proteins are upregulated in metastatic, castration-resistant prostate cancer (CRPC), a lethal form of the disease, compared to primary tumors and their normal counterparts. Disrupting the METTL-NUP93 complex by genetic mutations of residues mediating this interaction abrogated proliferation and migration of CRPC cells. These results suggest that the METTL3-NUP93 complex may play an important role in PCa progression to castration resistance and the underlying mechanism is worthy of further investigation. Considering that both NPCs and m6A machinery control the nucleocytoplasmic transport of mRNAs, we performed fractionation RNA-seq to identify mRNA molecules whose nuclear abundance is changed upon knockdown of METTL3 and NUP93 in PCa cells. A large group of mRNAs were significantly retained in the nuclei of both knockdown cells, suggesting that METTL3 and NUP93 coregulate the nuclear export of these mRNAs. The mRNA cargos of the METTL3-NUP93 complex are preferentially methylated, allowing them to be selected by the complex via association with METTL3. Interestingly, a subset of these mRNA cargos is functionally involved in the biosynthesis of the sex hormone androgen that is well known to activate the main driver of PCa progression, androgen receptor (AR). Indeed, overexpression of the wild-type METTL3 and NUP93, but not the mutant forms interrupting the new mRNA transport pathway, increases intracellular androgen levels in PCa cells, upregulates the expression of AR target genes even without addition of the ligand and confers the androgen- independent growth of PCa cells, implying the development of hormone-refractory phenotypes. All these effects can be alleviated by the pharmacological inhibitors of the steroidogenic enzymes encoded by the target mRNAs of the METTL3-NUP93 complex. So, our hypothesis is: Upregulation of METTL3 and NUP93 promotes PCa progression by facilitating the nuclear export of mRNAs, a subgroup of which encode androgen biosynthesis enzymes that trig...