Fig 2: CircCRIM1 binds to IGF2BP1 protein. (A) Biotin-labeled sense or antisense circCRIM1 probes were used for RNA-protein pull-down against A549 cell lysates. Identification of proteins that interact with circCRIM1 via silver staining. Red arrows indicate the major differential band precipitated in A549 lysates. (B) Analysis pipeline was performed to identify proteins that interact with circCRIM1 according to mass spectrometry assay. (C) IGF2BP1/2/3 immunoblot analysis of the biotin-labeled sense and antisense circCRIM1 probes pull-down eluate from lysates of A549 cells. (D) RNA immunoprecipitation (RIP) assays in A549 cells using IGF2BP1/2/3 and IgG antibody. (E) The FLAG-IGF2BP1/2/3-enriched circCRIM1 relative to the IgG-enriched value was calculated via qRT-PCR. Statistics by unpaired t-test. (F) Schematic diagram of full-length or truncations FLAG-tagged of IGF2BP1protein. Wt-IGF2BP1 (full-length IGF2BP1), KH1-KH4 (from KH1 to KH4 domain), RRM-KH2 (from RRM1 to KH2 domain), KH3-KH4 (from KH3 to KH4 domain), RRM (from RRM1 to RRM2 domain). (G) RIP analysis for circCRIM1 enrichment in A549 cells transiently transfected with plasmids containing the indicated FLAG-tagged full-length or truncated constructs. Relative enrichment of endogenous circCRIM1 in FLAG-tagged full-length or truncated IGF2BP1 RIP was measured using qRT-PCR. Statistics by unpaired t-test. (H) Dual RNA-FISH and immunofluorescence staining assays indicating the co-localization of circCRIM1 (red) and IGF2BP1 (green), with nuclei staining with DAPI (blue). Data shown as mean ± SD; ns, not significant; *** p < 0.001.

Fig 3: IGF2BP1/m6A/HMGB1 axis regulates high glucose-induced vascular endothelial cells apoptosis via m6A-dependent manner.

Fig 4: Functional grouping of mRNAs in IMP-1 complexes from mouse mammary epithelial (EP) cells.A, the mRNAs in control IgG complexes were subjected to STRING analysis, which found no significant enrichment groups (defined as FDR > 0.05) except for a (relatively unique) group of histone mRNAs, and intriguingly, the mRNA for IMP-1 itself. B, the mRNAs in IMP-1-associated complexes showed functional enrichment for two groups of genes, glutathione metabolism genes (including GPX-2) and mRNAs for proteins targeted to mitochondria, important for mitochondrial ribosome function and the assembly of complex 1 in the electron transport chain. FDR, false discovery rate; GPX-2, glutathione peroxidase; IgG, immunoglobulin G; IMP-1, insulin-like growth factor 2 mRNA-binding protein-1; STRING, Search Tool for the Retrieval of Interacting Genes/Proteins.

Fig 5: METTL3 regulates the translation of HDAC6 via YTHDF3. A) m6A peak was enriched in CDS of HDAC6 genes from m6A RIP‐seq data. B) m6A RIP‐qPCR analysis of HDAC6 mRNA in WT or METTL3 Mut/‐ HeLa cells. C) The relative mature‐mRNA levels of HDAC6 in METTL3‐knockdown HeLa or SiHa cells were measured. D) After treating WT or METTL3 Mut/− HeLa cells with 50 µg ml−1 CHX for the indicated time, the expression of HDAC6 was detected by western blot analysis (left) and quantitatively analyzed (right). E) The translation efficiency of endogenous HDAC6 was checked by normalizing HDAC6 protein levels to the corresponding mRNA abundance. F) WT or METTL3 Mut/− HeLa cells were transfected with pmirGLO‐HDAC6‐CDS‐WT reporter for 24 h, respectively. The protein, mRNA, and translation efficiency were determined. G) The polysome profiling of WT or METTL3 Mut/− HeLa cells was analyzed. H) The mRNA levels of HDAC6 in non‐translational fraction (< 40S), translation initiation fraction (including 40S, 60S ribosomes, 80S monosomes) and polysome fractions in WT or METTL3 Mut/− HeLa cells were analyzed by qRT‐PCR. I) RIP‐qPCR analysis of HDAC6 mRNA in HeLa WT cells using antibodies against YTHDF1–3, IGF2BP1–3, YTHDC2, and HNRNPA2B1. J) Binding between YTHDF3 and HDAC6 mRNA in WT or METTL3 Mut/− HeLa cells was detected by RIP‐qPCR. K) Expression levels of HDAC6 in WT or METTL3 Mut/− HeLa cells silencing YTHDF3 were detected by western blot assay. Data are presented as mean ± SD from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant, by Student's t‐test between two groups and by one‐way ANOVA followed by Bonferroni's test for multiple comparisons.