Fig 2: KIAA1429/VIRMA depletion reduces the proliferation, migration, and invasion of BCa cells A–F(A, C, E) RT–qPCR analysis (n = 3, biological replicates) and immunoblotting of KIAA1429 levels in MCF10A (A), triple‐negative MDA‐MB‐157 cells (C), or triple‐negative MDA‐MB‐231 cells (E) expressing control or two KIAA1429‐targeting shRNAs. **P < 0.01, ***P < 0.001. (B, D, F) MTS cell proliferation assay of MCF10A cells (B), triple‐negative MDA‐MB‐157 cells (D), or triple‐negative MDA‐MB‐231 cells (F) expressing control shRNA and or shKIAA1429. *P < 0.05, **P < 0.01, ***P < 0.001. Representative blots are shown. n = 3, biological replicates. Error bars show mean ± standard error (SEM). P‐values were calculated by Student's t‐test.GRepresentative images (left) and quantification (right) of a cell migration assay of control and KIAA1429 knockdown MDA‐MB‐231 cells. **P < 0.01. n = 3, biological replicates. Scale bar = 100 μm. Error bars show mean ± standard error (SEM). P‐value was calculated by Student's t‐test.HRepresentative images of three‐dimensional (3D) cell invasion assay of control and KIAA1429 knockdown MDA‐MB‐231 cells on Days 2 and 4 after plating. n = 3, biological replicates. Scale bar = 215 px.IGrowth of control and KIAA1429 knockdown MDA‐MB‐231 cells inoculated subcutaneously in immunodeficient NSG mice. ****P < 0.0001. n = 5 for each randomized mouse group. Error bars show mean ± standard error (SEM). Two‐way ANOVA was used to calculate P‐values.J–M(J, L) RT–qPCR analysis of METTL3 mRNA (J) or METTL14 (L) mRNA levels in control and KIAA1429 knockdown MDA‐MB‐231 cells. *P < 0.05, **P < 0.01. n = 3, biological replicates. Error bars show mean ± standard error (SEM). P‐values were calculated by Student's t‐test. (K, M) MTS cell proliferation assay of control and METTL3 knockdown (K) or METTL14 knockdown (M) MDA‐MB‐231 cells. *P < 0.05, **P < 0.01. n = 3, biological replicates. The error bars show the mean ± standard error (SEM). P‐values were calculated by Student's t‐test. See also Fig EV2.

Fig 3: KIAA1429/VIRMA promotes BCa proliferation and migration by stabilizing HAS2 mRNA expression in the cytosolIn normal breast epithelial cells, KIAA1429/VIRMA and the m6A methyltransferase complex are mainly located in the nucleus where it modifies RNA transcripts by depositing m6A. m6A‐modified mRNAs are then transported to the cytosol where they are either degraded or stabilized in an m6A‐dependent manner depending on the interaction with specific m6A reader proteins. In the present study, we show that in BCa cells, KIAA1429/VIRMA expression is upregulated and mislocalized to the cytosol where it interacts with IGF2BP3 and stabilizes m6A‐modified HAS2 mRNA, leading to enhanced cell proliferation and migration.

Fig 4: KIAA1429/VIRMA loss leads to reduced migration and proliferation in triple‐negative breast cell lines (related to Fig 2) mRNA levels of KIAA1429/VIRMA expression in shControl, shKIAA1429‐724, shKIAA1429‐727, shKIAA1429‐723, shKIAA1429‐725, and shKIAA1429‐726 MDA‐MB‐231 breast cancer cells. **P < 0.01; ***P < 0.001. n = 3, biological replicates. The error bars show the mean ± standard error (SEM). P‐values were calculated by Student's t‐test.Cell proliferation of shControl and shKIAA1429 293T cells. n = 3, biological replicates. The error bars show the mean ± standard error (SEM).mRNA levels of METTL3 expression in shControl, shMETTL3‐714, and shMETTL3‐717 MCF10A cells. ***P < 0.001. n = 3, biological replicates. The error bars show the mean ± standard error (SEM). P‐values were calculated by Student's t‐test.Cell proliferation of shControl and shMETTL3 MCF10A cells. n = 3, biological replicates. The error bars show the mean ± standard error (SEM).mRNA levels of METTL14 expression in shControl, shMETTL14‐933, and shMETTL14‐936 MCF10A cells. **P < 0.01. n = 3, biological replicates. The error bars show the mean ± standard error (SEM). P‐values were calculated by Student's t‐test.Cell proliferation of shControl and shMETTL14 MCF10A cells. n = 3, biological replicates. The error bars show the mean ± standard error (SEM).Control (shControl) and KIAA1429/VIRMA KD (shKIAA1429‐1, shKIAA1429‐2) MDA‐MB‐231 cells formed tumors for the experiments shown in Fig 2I. The length of all the tumors is less than 2 cm. Scale bar = 1 cm.Tumor growth in individual NOD‐scid IL2Rgammanull (NSG) mice for the experiments shown in Fig 2I. Each line shows data for one mouse.Upper: mRNA levels of KIAA1429 in nontargeting control (NTC) and KIAA1429/VIRMA KO MDA‐MB‐231 cells. *P < 0.05. n = 3, biological replicates. The error bars show the mean ± standard error (SEM). Down: Protein levels of KIAA1429 in nontargeting control (NTC) and KIAA1429/VIRMA KO MDA‐MB‐231 cells. Representative blots are shown. n = 3, biological replicates. P‐values were calculated by Student's t‐test.Cell proliferation of NTC and KIAA1429 KO (sg1, sg2) MDA‐MB‐231 cells. *P < 0.05; **P < 0.01. n = 3, biological replicates. The error bars show the mean ± standard error (SEM). P‐values were calculated by Student's t‐test.Representative images of cell migration of shControl and shKIAA1429 MDA‐MB‐157 breast cancer cells. Scale bar = 215 px.

Fig 5: KIAA1429/VIRMA depletion reduces m6A methylation and the stability of HAS2 mRNA in BCa cells Gene Ontology (GO) analysis of genes significantly downregulated (log2 fold‐change < −1) in MDA‐MB‐231 cells expressing KIAA1429/VIRMA‐targeting sgRNAs or nontargeting control (NTC) sgRNA. GO categories related to cell proliferation or migration are indicated by red boxes.Heatmap of genes significantly downregulated (log2 fold‐change < −1) in MDA‐MB‐231 cells expressing KIAA1429/VIRMA‐targeting sgRNAs or NTC sgRNA. The list shows the top downregulated genes associated with cell proliferation or migration.Representative m6A blotting of whole cell extracts of RNA from control (NTC) or KIAA1429 KO MDA‐MB‐231 cells. (Left) RNA gels indicate equal amount of RNAs used in m6A blots with UV illumination. (Right) m6A blots of control and KIAA1429 KO cells show a reduction of m6A modification in KIAA1429‐depleted cells. n = 3, biological replicates.Motif analysis of MeRIP‐seq data from MDA‐MB‐231 cells expressing control or KIAA1429‐targeting shRNA. Arrows indicate m6A‐modified adenosine.Venn diagrams showing overlap between mRNAs with reduced m6A content and mRNAs with reduced expression level in MDA‐MB‐231 cells expressing KIAA1429‐targeting shRNAs compared with control shRNA cells.m6A profile of HAS2 mRNA in MDA‐MB‐231 cells expressing KIAA1429‐targeting shRNAs compared with control MDA‐MB‐231 cells visualized in Integrated Genome Browser tracks. Red color indicates m6A profile in control group, green color indicates m6A profile in KIAA1429 knockdown cells, and blue bar below each track indicated called peaks.RT–qPCR analysis of KIAA1429, HAS2, and JUN mRNA levels in MDA‐MB‐231 cells expressing control or KIAA1429‐targeting shRNA. *P < 0.05, **P < 0.01. n = 3, biological replicates. Error bars show mean ± standard error (SEM). P‐values were calculated by Student's t‐test.RT–qPCR analysis of HAS2 mRNA in MDA‐MB‐231 cells expressing control or KIAA1429‐targeting shRNA after incubation with 5 μg/ml actinomycin D (ActD) for the indicated times. *P < 0.05. n = 3, biological replicates. Error bars show mean ± standard error (SEM). P‐value was calculated by Student's t‐test.Western blot analysis of KIAA1429, HAS2 and phosphorylated AKT (pAKT Ser473) in MDA‐MB‐231 cells expressing control or KIAA1429‐targeting shRNA following incubation with 10 ng/ml epidermal growth factor (EGF) for 5 or 30 min. GAPDH was probed as a loading control. Representative blots are shown. n = 3, biological replicates.Immunoblot analysis of KIAA1429 and HAS2 in MDA‐MB‐231 formed tumors grown on immunodeficient NOD‐scid IL2Rgammanull (NSG) mice. GAPDH was probed as a loading control. Representative blots are shown. n = 3, biological replicates. See also Fig EV3, Datasets EV1 and EV2.