Fig 2: Bcas2 may be involved in the expression and alternative splicing of CSR‐related genes in hyper IgM. (A) Pearson's correlation analysis shows the coefficient between two replicates in the sample of the normal people and the patient in PBMC proteomics. (B) Circos representing the correlation between different proteins and normal people or the HIGM1 patient; with a cluster heatmap of several proteins related to antibody formation. Red indicates a higher expression level, while blue indicates a lower expression level. (C) Volcano map displaying the distribution of differentially expressed proteins from proteomics data. The abscissa in the figure represents the protein fold change in PBMC of normal human and Hyper IgM patient. |FoldChange| ≥ 1.5. Padj ≤ 0.05. The ordinate indicates the significance of gene expression differences between the HIGM patient and normal subjects. Upregulated genes are shown as red dots, and downregulated genes are shown as blue dots. (D) Scatter plot of differentially expressed proteins. The abscissa is the protein indexes, and the ordinate is the −log10 (p value) of the differentially expressed protein. Red indicates a higher expression level above the threshold with the −log10 (p value) equals 1.5. (E) Network showing GO enrichment analyses of differentially expressed proteins of Hyper IgM patient. (F) Differentially expressed proteins of Hyper IgM patient from PBMC proteomics. All the relative expression levels of proteins in normal people were normalized to 1.

Fig 3: Bcas2 regulates mRNA abundance in activated‐B cells. (A) The expression of Bcas2 is shown as coverage tracks in LPS+IL4 stimulated WT and Bcas2‐cKO B cells. (B) Pearson correlation analysis shows the coefficients between two replicates of WT and Bcas2‐cKO mice in the RNA‐seq data. (C) Volcano map displaying the distribution of differentially expressed genes from RNA‐seq data. The abscissa in the figure represents the gene fold change in LPS+IL4 stimulated WT and Bcas2‐cKO B cells. |FoldChange| ≥ 1.5. Padj ≤ 0.05. The ordinate indicates the significance of gene expression differences between LPS+IL4 stimulated WT and Bcas2‐cKO B cells. Upregulated genes are shown as red dots, and downregulated genes are shown as green dots. (D) Cluster heatmap of differentially expressed genes. The abscissa is the genotype, and the ordinate is the normalized FPKM (fragments per kilobase million) value of the differentially expressed gene. Yellow indicates a higher expression level, while blue indicates a lower expression level. (E) Network showing GO enrichment analyses of differentially expressed genes. (F) Heatmap of CSR‐associated, DNA recombination and repair‐associated, and Bcas2‐binding gene expression. (G) The expression of CSR‐associated, DNA recombination and repair‐associated, and Bcas2‐binding genes in LPS+IL4 stimulated WT and Bcas2‐cKO B cells. The RT‐qPCR data were normalized to gapdh. The value in the WT group was set as 1.0, and the relative value in the Bcas2‐cKO group is indicated (n = 3). Unpaired Student's t test determined significance and exact p value. The points and error bars represent the mean ± SEM. (H) RNA‐seq data represent the expression of CSR‐associated, DNA recombination and repair‐associated, and Bcas2‐binding genes.

Fig 4: Bcas2 recruits AS‐related proteins to modulate AS in B cell. (A) Silver‐stained gel of Bcas2 and control immuno‐precipitates from splenic B cells. (B) Scatter plot of protein scores demonstrates significant differences in proteins between two groups of parallel mixed samples. (C) IP experiment was performed in spleen B cells extracts after LPS stimulation. (D) Network showing GO enrichment analyses of Bcas2‐binding proteins. (E) Co‐immunostaining and intensity scan map of CH12 cell was performed using Bcas2 and Bcas2‐binding proteins (SRSF7 and DHX15) antibodies. DNA was stained with DAPI. Scale bar, 1 µm. (F) Quantitative PCR analysis and western blot of B cell mRNA of NC, siBcas2, siSRSF7 and siS+B. Bcas2 and SRSF7 expression levels were normalized to gapdh transcripts and the NC group in quantitative PCR analysis (n = 3, mean ± SD). (G) Flow cytometric analysis for CSR in CH12 cell lines of NC, siBcas2, siSRSF7 and siS+B after CIT stimulation. (H) Quantification of CSR shown in (G). (I) Alternative splicing of Bcas3 and Cd72 in CH12 cell lines of NC, siBcas2, siSRSF7 and siS+B was analyzed by RT‐PCR (n = 3). The ratios of inclusion (Incl) to exclusion (Excl) are shown accordingly. (J) Model figure of CC1 and CC2 sequence location in Bcas2 protein. (K) Immunoprecipitation of Bcas2 flag and SRSF7‐HA. (L) Conservation of amino acid sequences in protein interaction regions across different species is demonstrated. (M) Flow cytometric analysis for CSR in CH12 cells with the deletion of CC1 and CC2 sequence of Bcas2 and SRSF7 knockdown. (N) Quantification of CSR shown in (M). Each symbol represents CH12 cell cultures (n = 3, mean ± SD). (O) The alternative splicing events in CH12 cells with the deletion of CC1 and CC2 sequence of Bcas2 and SRSF7 knockdown.

Fig 5: Bcas2 directly binds mRNA to regulate mRNA alternative splicing. (A) Venn diagram showing the correlation among down regulated, upregulated, alternatively spliced, and Bcas2‐binding genes. (B) Integrated RNA‐seq and CLIP‐seq analyses show that Bcas2 binds to genes undergoing either up‐ or down‐regulation of various types of alternative splicing events. (C) Alternative splicing of Cd72, Msi2 and Rps6ka5 in the spleen of LPS+IL4 stimulated WT and Bcas2‐cKO B cells was analyzed by RT‐PCR (n ≥ 3). The ratios of inclusion (Incl) to exclusion (Excl) are shown accordingly. Analysis of CSR‐associated, DNA recombination and repair‐associated, and Bcas2‐binding gene expression and exon‐exon junctions. The Bcas2‐binding peaks of meiosis‐related gene transcripts are shown. Purple arrow, direction of transcription. F, forward primers; R, reverse primers; D, Co‐immunostaining of Ctrl and cKO was performed using target protein (Cd72, Msi2 and Rps6ka5) and B220 antibodies from CH12 cell lines. DNA was stained with DAPI. Scale bar, 1 µm. (E) Schematic diagram of the construction of Cd72 targeted mutations (location of motifs significantly bound by Bcas2). (F) Quantitative PCR analysis of OE, EV and KD B cells. Bcas2 expression levels were normalized to gapdh transcripts and the EV group in quantitative PCR analysis (n = 3, mean ± SD). (G) Western blot analysis of EV and KD B cells on Bcas2 expression levels. (H) Alternative splicing of Cd72 in WT and mutant CH12 cell lines of OE, EV and KD was analyzed by RT‐PCR (n = 3). The ratios of inclusion (Incl) to exclusion (Excl) are shown accordingly. (I) Flow cytometric analysis for CSR in WT and mutant CH12 cell lines of OE, EV and KD after CIT stimulation. (J) Quantification of CSR shown in (I). Each symbol represents CH12 cell cultures (n = 3, mean ± SD). OE, overexpression; EV, empty vector; KD, knockdown.