USP25 Facilitates METTL3 Deubiquitination To Promote Breast Cancer Cells Migration And Invasion

N6-methyladenosine(m6A)is a chemical modification present in multiple RNA species,being most abundant in mRNAs.M6A modification has revealed their roles throughout the life cycle of RNA,it regulates several facets of RNA processing or metabolism,including alternative splicing,export,stability,and translation,that affects embryonic development and cell differentiation,and altered m6A homeostasis is linked to cancer.The m6A modification is deposited by the m6A methyltransferase complex and can be removed by m6A demethylases.The fates of m6A-modified RNA are determined by the functions of binding proteins.M6A modification is catalysed by METTL3 and strongly enriched in the 3' untranslated region of mRNAs at sites close to the stop codon.In addition,the cytoplasmic METTL3 can also enhance mRNA translation.However,the characteristics of METTL3 in activation and post-translational modification(PTM)is seldom understood,such as ubiquitylation.Ubiquitination affects proteins in many ways:it can mark them for degradation via the proteasome,alter their cellular location,affect their activity,and promote or prevent protein interactions.Here we find that METTL3 interacts with deubiquitinating enzyme USP25 in a large-scale DUB screen performed in HEK-293 cells by Co-Immunoprecipitation.Moreover,the USP domain of USP25 directly interacts with the gate-loop 1 and interface loop of METTL3.METTL3 cellular co-localization with USP25 mainly in the cytoplasm of breast cancer cells MDA-MB-231 by immunofluorescence microscopy.METTL3 ubiquitination is significantly diminished by USP25 in vivo and intro.On the other hand,USP25C178S,the catalytic inactive USP25 mutant with a mutation at the core enzymatic domain,lost the ability to reverse METTL3 ubiquitination.Conversely,downregulation of USP25 in HEK-293T cells or knockout of USP25 in mouse embryonic fibroblast cells increase METTL3 ubiquitination.Further studies show that USP25 specifically cleaves Lys48(K48)-linked polyubiquitin from METTL3.Since USP25 is an ubiquitin-specific protease,it is possible that USP25 could function to stabilize METTL3.Coexpression with USP25 significantly increased the protein levels of METTL3.Knockdown or knockout USP25 decreased METTL3 protein levels with no effect on METTL3 mRNA levels,while reconstitution with shRNA-resistant USP25 restored METTL3 levels.In addition,the decrease in METTL3 levels could be reversed by proteasome inhibitor MG 132,suggesting that USP25 regulates METTL3 levels in a proteasome-dependent manner.The protein half life of METTL3 significantly shorten in cells stably expressing USP25 shRNA or USP25-/-MEF cells when treated with cycloheximide.Interestingly,downexpression of USP25 significantly decreases the protein half-life of METTL3,but does not alter its m6A methytransferase activity.Afterwards,we identify USP25 only affects the stability of cytoplasmic METTL3,but not nuclear METTL3.In addition,knockdown of USP25 notably depresses the protein translation of EGFR,which can be rescued by overexpress the wide-type METTL3 and the catalytic mutant of METTL3,but not the A155P mutant of METTL3.Finally,knockdown USP25 suppresses breast cancer cell migration and invasion with METTL3.Collectively,our study finds the first DUB of METTL3,USP25 regulates the stability of cytoplasmic METTL3 by cleaves its Lys48(K48)-linked polyubiquitin.USP25 facilitates EGFR translation through METTL3,USP25 or/and METTL3 depletion significantly suppressed the ability of MDA-MB-231 breast cancer cells migration and invasion.These fundings might contribute new therapeutic treatments for cancer.