eIF4A1 and eIF4A2 play critical roles during early B-cell development

We first examined the expression of eIF4A1 and eIF4A2 during B-cell development in the bone marrow and in mature B cells activated by various stimuli mimicking B-cell receptor stimulation (anti-IgM), T-cell help (anti-CD40 and anti-CD40 + IL-4), and T-cell-independent signaling (LPS and LPS + IL-4). As shown in Fig. 1A–C, eIF4A1 mRNA expression was high in Fraction C (Fr. C), diminished afterwards, and strongly induced upon B cell activation. In contrast, eIF4A2 mRNA expression gradually increased during B cell development and reached the highest level in mature B cells recirculated back to the bone marrow (Fr.F). Upon B cell activation, eIF4A2 mRNA and protein expression was upregulated to various degrees by 24 h but decreased by 48 h (Fig. 1B, C). The dynamic changes in the expression of eIF4A1 and eIF4A2 during B-cell development and activation suggest important roles of this family in B cells. To investigate the biological function of eIF4A1 and eIF4A2, we generated loxP-site flanked alleles of these two genes (Supplementary Fig. 1A–D) and crossed the mutant mice with Mb1Cre mice in which the CD79a gene was replaced with a codon-optimized Cre recombinase gene [21]. The CD79a locus turns on Cre expression and drives efficient deletion of loxP site-flanking alleles at an early developmental stage of the B-cell lineage. Both Eif4a1fl/fl;Mb1Cre and Eif4a2fl/fl;Mb1Cre mice appeared healthy but presented few B cells in the spleen and peripheral lymph nodes (Supplementary Fig. 2). Loss of peripheral B cells may be caused by defective B-cell development. Thus, we examined B-cell development in the bone marrow. Consistent with high eIF4A1 expression in Fr.C. (Fig. 1A), Eif4a1fl/fl;Mb1Cre mice presented a progressive and drastic decrease in the number of Fr.B. and Fr.C. cells and a complete lack of B lineage cells after Fr.C. (Fig. 1D–G). Eif4a2fl/fl;Mb1Cre mice exhibited similar impairments in B cell development, but the decrease did not manifest until the Fr.D, accompanied by a slight reduction in the percentage of Fr.C cells (Fig. 1H–K). Therefore, both eIF4A1 and eIF4A2 play critical roles during early B cell development, but at different developmental stages.

Fig. 1

eIF4A1 and eIF4A2 are required for early B cell development. A RT‒qPCR analysis of eIF4A1 and eIF4A2 mRNA expression in bone marrow B lineage cells. B, C RT‒qPCR B and immunoblot C analysis of eIF4A1 and eIF4A2 mRNA and protein expression in naïve B cells activated with various stimuli for the indicated amounts of time. D–K Flow cytometry analysis of B lineage cells in the bone marrow of Eif4a1fl/fl, Eif4a1fl/fl;Mb1Cre D–G and Eif4a2fl/fl, Eif4a2fl/fl;Mb1Cre H–K mice. D, E, F, H, I, J Representative FACS plots. G, K Bar graphs summarizing cell numbers. Each symbol represents an individual mouse. The error bars represent the standard errors of the means (SEMs). ns, not significant; *p < 0.05, **p < 0.01 and ***p < 0.001. The data shown are representative of three independent experiments. In G, K, the data represent the combination of two independent experiments

eIF4A1 and eIF4A2 are indispensable for the T-cell-dependent antibody response

The lack of peripheral B cells in Eif4a1fl/fl;Mb1Cre and Eif4a2fl/fl;Mb1Cre mice prevented the study of the immune functions of eIF4A1 and eIF4A2. Therefore, we crossed Eif4a1fl/fl and Eif4a2fl/fl mice with CD19Cre mice, in which expression of the Cre recombinase is under the control of the CD19 locus [22, 23]. Cre expression is turned on at Frs. B and C in CD19Cre mice, with a deletion efficiency of about 33% in bone marrow B cells and 90–97% in mature B cells of the periphery [22, 23]. This allows bypassing of early B-cell developmental defects in Eif4a1fl/fl;Mb1Cre and Eif4a2fl/fl;Mb1Cre mice. B-cell development was normal in Eif4a1fl/fl;CD19Cre and Eif4a2fl/fl;CD19Cre mice. The proportions and numbers of various B-cell subsets in the spleen and peripheral lymph nodes of Eif4a1fl/fl;CD19Cre and Eif4a2fl/fl;CD19Cre mice were also similar to that of wild type mice (Supplementary Fig. 3).

To investigate the roles of eIF4A1 and eIF4A2 in the humoral immune response, Eif4a1fl/fl;CD19Cre and Eif4a2fl/fl;CD19Cre mice were immunized with ovalbumin (OVA) precipitated in alum accompanied with lipopolysaccharide (LPS) (termed OVA/alum/LPS). Flow cytometry analysis of splenocytes from immunized mice showed that the percentage and number of GCB and plasma cells were significantly reduced in Eif4a1fl/fl;CD19Cre and Eif4a2fl/fl;CD19Cre mice (Fig. 2A, B, F, G). Furthermore, we examined the antigen-specific antibody response by immunizing Eif4a1fl/fl;CD19Cre and Eif4a2fl/fl;CD19Cre mice with 4-hydroxy-3-nitrophenyl hapten conjugated to ovalbumin (NP-OVA) precipitated in alum (termed NP-OVA/alum). As shown in Fig. 2C–E, H–J, NP-specific total IgG1 (anti-NP30 IgG1) and high-affinity IgG1 (anti-NP5 IgG1) were drastically decreased in both Eif4a1fl/fl;CD19Cre and Eif4a2fl/fl;CD19Cre mice, accompanied by a slight reduction in NP-specific IgM antibodies. Taken together, our results demonstrate that both eIF4A1 and eIF4A2 are indispensable for the T-cell-dependent B-cell response, including GCB formation, plasma cell differentiation, and antibody production.

Fig. 2

eIF4A1 and eIF4A2 are indispensable for the T-cell-dependent B cell response. A, B, F, G Flow cytometry analysis of GCB (CD38-CD95+) A, F and plasma cells (B220low/-CD138+) B, G in the spleens of Eif4a1fl/fl (n = 10), Eif4a1fl/fl;CD19Cre (n = 7) A, B and Eif4a2fl/fl (n = 10), and Eif4a2fl/fl;CD19Cre (n = 6) F, G mice on day 7.5 after immunization with OVA/alum/LPS. Left, representative FACS plots; right, summary of the percentage and number of GCB and plasma cells. C, D, E, H, I, J Eif4a1fl/fl (n = 8), Eif4a1fl/fl;CD19Cre (n = 6), Eif4a2fl/fl (n = 7), and Eif4a2fl/fl;CD19Cre (n = 6) mice were immunized with NP-OVA/Alum. Serum concentrations of NP-specific IgM C, H and IgG1 D, E, I, J antibodies at the indicated time points were determined by ELISA. Each symbol represents an individual mouse. The error bars represent the standard errors of the means (SEMs). ns, not significant; *p < 0.05, **p < 0.01 and ***p < 0.001. The data shown are representative of three independent experiments

Differential roles of eIF4A1 and eIF4A2 in T-cell-independent antibody responses

We further investigated the roles of eIF4A1 and eIF4A2 in T-cell-independent (TI) antibody responses. Eif4a1fl/fl;CD19Cre and Eif4a2fl/fl;CD19Cre mice were immunized with 4-hydroxy-3-nitrophenyl hapten-conjugated LPS (NP-LPS), a type 1 TI (TI-1) antigen that activates B cells through both the NP-specific B-cell receptor (BCR) and Toll-like receptor 4 (TLR4). As shown in Fig. 3A–D, B-cell-specific deletion of either eIF4A1 or eIF4A2 resulted in drastic reductions in the percentage and number of plasma cells, as well as the titers of NP-specific IgM antibodies. We then immunized these mice with NP-Ficoll, a type 2 TI (TI-2) antigen that consists of a highly repetitive surface structure (NP) conjugated to a polysaccharide (Ficoll). NP-Ficoll activates NP-specific B cells by crosslinking many BCRs, resulting in proliferation, plasma cell differentiation, and antibody production. While B-cell-specific deletion of eIF4A1 had no obvious effect on the production of NP-specific IgM and IgG3 antibodies (Fig. 3E, F), deletion of eIF4A2 caused significant reductions in both NP-specific IgM and IgG3 antibodies (Fig. 3G, H). Therefore, eIF4A1 is required only for the TI-1 antibody response, while eIF4A2 is required for both the TI-1 and TI-2 antibody responses.

Fig. 3

eIF4A2 controls both the TI-1 and TI-2 B-cell responses, whereas eIF4A1 is required for the TI-1 response. A, C Flow cytometry analysis of plasma cells in the spleens of Eif4a1fl/fl (n = 6), Eif4a1fl/fl;CD19Cre (n = 6) A and Eif4a2fl/fl (n = 4), and Eif4a2fl/fl;CD19Cre (n = 5) C mice on day 3 after NP‒LPS immunization. Left, representative FACS plots; right, summary of the percentage and number of plasma cells. B, D Serum concentrations of the NP-specific IgM antibody were determined via ELISA at the indicated time points after NP‒LPS immunization in Eif4a1fl/fl (n = 6), Eif4a1fl/fl;CD19Cre (n = 7) B, Eif4a2fl/fl (n = 10), and Eif4a2fl/fl;CD19Cre (n = 7) D mice. E–H ELISA analysis of NP-specific IgM E, G and IgG3 F, H in Eif4a1fl/fl (n = 6), Eif4a1fl/fl;CD19Cre (n = 5) E, F and Eif4a2fl/fl (n = 8), and Eif4a2fl/fl;CD19Cre (n = 6) G, H mice after NP-Ficoll immunization. Each symbol represents an individual mouse. The error bars represent the standard errors of the means (SEMs). ns not significant; *p < 0.05, **p < 0.01 and ***p < 0.001. The data shown are representative of three independent experiments

eIF4A1 and eIF4A2 control B cell proliferation

We wondered whether reduced GCB formation, plasma cell differentiation, and antibody production resulting from B-cell-specific deletion of eIF4A1 and eIF4A2 were caused by impaired B-cell activation and proliferation. B cells were purified from Eif4a1fl/fl;CD19Cre and Eif4a2fl/fl;CD19Cre mice, stimulated with LPS, and examined for activation and proliferation. As shown in Fig. 4C, F, the absence of either eIF4A1 or eIF4A2 had no obvious effect on B-cell activation, as indicated by the normal upregulation of activation markers CD83, CD86, and CD69. Instead, it severely impaired B-cell proliferation by blocking the G1/S transition during cell cycle progression (Fig. 4D, E, G, H). Taken together, our findings show that eIF4A1 and eIF4A2 are dispensable for activation of B cells, but essential for their cell cycle progression and proliferation.

Fig. 4

eIF4A1 and eIF4A2 control B cell proliferation. A, B Immunoblot analysis of eIF4A1 and eIF4A2 protein expression in B cells purified from Eif4a1fl/fl, Eif4a1fl/fl;CD19Cre A, Eif4a2fl/fl, and Eif4a2fl/fl;CD19Cre B mice and stimulated with LPS for the indicated amounts of time. C, F Flow cytometry analysis of activation marker expression on B cells stimulated with LPS for the indicated amounts of time. D, G B cells were labeled with CellTrace Violet (CTV), stimulated with LPS for 3 days, and analyzed by flow cytometry for cell division. E, H B cells were stimulated with LPS for 48 h, treated with EdU for 2 h, and analyzed by flow cytometry for cell cycle progression. The error bars represent the standard errors of the means (SEMs). ns, not significant; *p < 0.05, **p < 0.01 and ***p < 0.001. The data shown are representative of three independent experiments

eIF4A2 controls the biogenesis of the 40S ribosome subunit

Previous studies have shown that eIF4A1 and eIF4A2 are components of the eIF4F complex and play critical roles in translation initiation by unwinding secondary structures in the 5’UTRs of mRNAs. We therefore examined the impact of their absence on global translation in B cells. Naïve B cells from Eif4a1fl/fl;CD19Cre and Eif4a2fl/fl;CD19Cre mice were activated with LPS for 24 hours. The cytoplasmic contents were harvested and separated in a sucrose density gradient (Fig. 5A). As shown in Fig. 5B, there was an overall reduction in heavy polysome fractions accompanied by a significant accumulation of 80S monosome fractions in Eif4a1-deficient B cells, indicating a decrease in the global translation rate. Strikingly, Eif4a2 deficiency resulted in a drastic reduction in the 40S fraction and an overall reduction in the heavy polysome fractions, while the 80S monosome fraction was not much affected (Fig. 5C). The polysome profiling results were further corroborated by immunoblot analysis of ribosomal proteins in polysome fractions. As shown in Fig. 5D, E, while Eif4a1-deficient B cells exhibited an overall reduction of ribosomal proteins in 80S and polysome fractions, the small ribosome subunit protein RPS6 was almost absent from the 40S fractions of Eif4a2-deficient B cells. This, together with the drastic reduction in 40S fractions in the polysome profiles of Eif4a2-deficient B cells (Fig. 5C), prompted us to hypothesize that eIF4A2 controls RPS protein expression and biogenesis of the 40S subunit of ribosome. 40S ribosome contains 18S rRNA and 33 ribosomal proteins (RPs). Eukaryotic ribosome assembly starts with the transcription of a polycistronic 47S rRNA precursor in the nucleolus. Emerging rRNA stretches are quickly bound by early assembly RPs and ribosome biogenesis factors (RBFs), giving rise to the 90S precursor particle, which is subsequently separated into pre-40S and pre-60S particles through a critical endonucleolytic pre-rRNA cleavage event. Both particles undergo numerous maturation steps, nuclear export, and final assembly in the cytoplasm, giving rise to mature and translationally competent 40S and 60S ribosome subunits [24]. We quantified RPS and RPL proteins in Eif4a1- and Eif4a2-deficient B cells before and after LPS stimulation by label-free quantitative mass spectrometry (LFQ-MS). As shown in Fig. 5F, G, LPS stimulation substantially elevated the expression of RPS and RPL proteins. While Eif4a1 deficiency had marginal effect on Eif4a2 upregulation, Eif4a2 deficiency significantly impaired upregulation of RPS proteins and, to a lesser degree, RPL proteins. Taken together, these data suggest that eIF4A1 controls global translation, while eIF4A2 regulates LPS-induced upregulation of RPS protein expression and biogenesis of the 40S ribosome subunit.

Fig. 5

eIF4A2 is required for 40S ribosome biogenesis. A Outline of polysome profiling experiments. B, C Polysome profiling graphs of Eif4a1fl/fl, Eif4a1fl/fl;CD19Cre B, Eif4a2fl/fl, Eif4a2fl/fl;CD19Cre C B cells stimulated with LPS for 24 h. D, E Immunoblot analysis of eIF4A1, eIF4A2, and ribosomal proteins in the indicated sucrose gradient fractions of B-cell lysates. Red underlines indicate the absence of RPS6 protein in the 40S fractions of Eif4a2-deficient B cells E. F, G Heatmap of RPS F and RPL G proteins in WT (C57BL/6 J), Eif4a1fl/fl;CD19Cre and Eif4a2fl/fl;CD19Cre B cells stimulated with LPS for the indicated durations. Two-way ANOVA was performed. The asterisks on the right side of each panel indicate the differences between WT and Eif4a1- or Eif4a2-deficient B cells at 24 hours after LPS stimulation F, G. *p < 0.05, **p < 0.01 and ***p < 0.001. The data shown are representative of three independent experiments. Each column represents an individual biological sample

eIF4A2 is required for 18S rRNA maturation

Lymphocyte activation is accompanied by a drastic increase in the number of ribosomes, translational output, and protein synthesis to sustain cellular growth and proliferation. In mammalian cells, ribosome biogenesis is controlled by two main mechanisms: rRNA synthesis and ribosomal protein expression. Immunoblot analysis of individual ribosomal proteins confirmed that the LPS-induced upregulation of RPS proteins was impaired in Eif4a2-deficient B cells (Figs. 5F, G, 6A). RNAseq analysis of the transcriptomes of Eif4a2fl/fl and Eif4a2fl/fl;CD19Cre B cells before and after LPS stimulation revealed little difference in the mRNA expression levels of RPS and RPL proteins between time points and genotypes (Fig. 6B, C), suggesting that eIF4A2 regulates RPS protein expression mainly through post-transcriptional mechanisms.

Fig. 6

eIF4A2 is required for 18S rRNA maturation. A Immunoblot analysis of eIF4A2 and ribosomal proteins in Eif4a2fl/fl and Eif4a2fl/fl;CD19Cre B cells stimulated with LPS for the indicated amounts of time. B, C Heatmap of the mRNA levels of the RPS B and RPL C genes determined by RNA-seq analysis of Eif4a2fl/fl and Eif4a2fl/fl;CD19Cre B cells stimulated with LPS for the indicated amounts of time. D The 18S/28S rRNA ratio was quantified via the Agilent 5400 Fragment Analyzer System to measure the amounts of 18S and 28S rRNA in total RNA from Eif4a2fl/fl and Eif4a2fl/fl;CD19Cre B cells stimulated with LPS for the indicated amounts of time. E Eif4a2fl/fl and Eif4a2fl/fl;CD19Cre B cells were stimulated with LPS for 12 hours, treated with MG132 or DMSO for the indicated amounts of time, and analyzed by immunoblotting. F Scheme of 18S rRNA maturation. G, H Northern blot analysis of total RNA from B cells stimulated with LPS for 24 hours using ITS-29 G, U3, U6, U14, and U22 probes H. The error bars represent the standard errors of the means (SEMs). ns, not significant; *p < 0.05, **p < 0.01 and ***p < 0.001. The data shown are representative of three independent experiments

Previous studies have shown that ribosomal protein expression is mainly controlled by the rate of rRNA synthesis [25]. Ribosomal proteins are often synthesized at high levels beyond that required for ribosome subunit production. This is balanced by continual degradation of ribosomal proteins not assembled with rRNA. Indeed, the 18S/28S rRNA ratio in LPS-activated Eif4a2fl/fl;CD19Cre B cells was significantly less than that in Eif4a2fl/fl B cells (Fig. 6D), suggesting compromised biogenesis of 18S rRNA. In addition, treatment of LPS-stimulated B cells with MG132, a proteasome inhibitor, substantially restored the expression of RPS proteins in Eif4a2-deficient B cells (Fig. 6E). Taken together, these results show that impaired upregulation of RPS proteins in LPS-activated Eif4a2-deficient B cells is mainly caused by compromised biogenesis of 18S rRNA and subsequent degradation of RPS proteins not assembled with 18S rRNA.

This finding prompted us to examine the maturation of 18S rRNA, the RNA component of the 40S ribosome subunit, in eIF4A2-deficient B cells. 18S rRNA is generated from the 47S rRNA precursor through a complex series of endonucleolytic and exonucleolytic cleavage steps (Fig. 6F) [26]. Northern blot analysis of 47S rRNA processing intermediates showed significant accumulation of 34S rRNA, reduction of 20S and 18S-E, and decrease of mature 18S, but not 28S, rRNA (Fig. 6G and Supplementary Fig. 4). We investigated the expression of several small nucleolar RNAs that play critical roles in 18S rRNA biogenesis [27,28,29,30]. As shown in Fig. 6H, U3 expression was reduced in eIF4A2-deficient B cells, whereas the levels of U6, U14, and U22 were comparable between Eif4a2fl/fl and Eif4a2fl/fl;CD19Cre B cells. Taken together, these findings show that eIF4A2 controls 40S ribosome subunit biogenesis by regulating 18S rRNA maturation.

eIF4A1 controls the cell cycle through translational regulation of Gins4 and other genes

We next investigated the molecular mechanisms underlying eIF4A1 control of the cell cycle. B cells from Eif4a1fl/fl and Eif4a1fl/fl;CD19Cre mice were stimulated with LPS for 24 hours and analyzed by label-free quantitative mass spectrometry (LFQ-MS) and RNA-seq to elucidate the impact of eIF4A1-deficiency on the proteome and transcriptome, respectively (Fig. 7A). While RNAseq analysis identified 533 up-regulated and 547 down-regulated genes, mass spectrometry analysis revealed 163 up-regulated and 220 down-regulated proteins. Genes with protein or RNA levels downregulated in Eif4a1-deficient B cells were analyzed for KEGG pathway enrichment. Consistent with the critical role of eIF4A1 in controlling cellular proliferation (Fig. 4D, E), the cell cycle was the most significantly enriched molecular pathway among the genes downregulated in either protein or RNA level (Fig. 7B). Previous studies have demonstrated critical roles of eIF4A1 in controlling translation initiation [31]. Eif4a1-deficient B cells exhibited a decrease in the global translation rate (Fig. 5B). We reasoned that direct target genes of eIF4A1 should exhibit alterations in protein, but not mRNA, levels in Eif4a1-deficient B cells. We therefore compared the differentially expressed genes in RNA levels (RNA-DEGs) with the differentially expressed genes in protein levels (MS-DEGs) and identified 248 DEGs mainly altered at the protein level (Fig. 7C). Metascape analysis of these genes identified a gene set of “regulation of DNA-directed DNA polymerase activity” (Fig. 7D), which includes Gins1 and Gins4, proteins of the Cdc45-MCM-GINS (CMG) complex. The CMG complex functions in the DNA melting and unwinding steps as a component of replisome during DNA replication in mammalian cells [32]. We speculate that these Gins proteins are the main mediators of eIF4A1 control of the cell cycle. Immunoblot analysis showed that the Gins4 protein was significantly induced upon LPS stimulation of Eif4a1fl/fl B cells and that this induction was much less in the absence of eIF4A1 (Fig. 7E). eIF4A1 promotes cap-dependent translation initiation by unwinding secondary structures in the 5’UTR of mRNA [1]. Indeed, the 5’UTR of Gins4 mRNA harbors highly complicated secondary structures when compared to Actb mRNA. Moreover, the minimum free energy of Gins4 5’UTR secondary structures is −90.50 kcal/mol, which is three-fold lower than that of Actb 5’UTR (Fig. 7F), suggesting a critical role of eIF4A1 in unwinding secondary structures of the Gins4 5’UTR to promote translation initiation of this mRNA. We examined the distribution of Actb and Gins4 mRNAs in the sucrose gradient (Fig. 7G). While the distribution of Actb mRNA showed a slight shift from heavy to light polysomes in Eif4a1-deficient B cells, Gins4 mRNA showed a much greater shift toward 80S monosome and light polysome fractions, indicating a strong dependency of Gins4 mRNA on eIF4A1 for translation initiation.

Fig. 7

eIF4A1 controls the translation of Gins4 via 5’UTR. A Experimental outline of the RNAseq and LFQ-MS experiments. B KEGG pathway enrichment of downregulated proteins identified by LFQ-MS and downregulated mRNAs quantified by RNA-seq. C Venn diagram analysis of differentially expressed genes (DEGs) identified via RNA-seq (RNA-DEGs) and LFQ-MS (MS-DEGs). Cutoff, absolute Log2FoldChange greater than 0.5. D A total of 248 genes from C with only altered protein expression were analyzed via Metascape for GO enrichment, and the top ten terms were plotted. E Immunoblot analysis of eIF4A1, Gins1, and Gins4 in Eif4a1fl/fl and Eif4a1fl/fl;CD19Cre B cells stimulated with LPS for the indicated amounts of time. F RNA structures of Gins4 and Actb 5’UTRs were predicted via RNAfold. MFE, minimum free energy. G Distribution of Actb and Gins4 mRNAs in the sucrose gradient fractions of Eif4a1fl/fl and Eif4a1fl/fl;CD19Cre B cells stimulated with LPS for 24 hours. H Graphical outline of the luciferase reporter gene. I Immunoblot analysis of eIF4A1 protein expression in HEK293T cells transfected with scramble or Eif4a1 siRNA (left panel). Luciferase assay of the reporter gene harboring the Actb or Gin4 5’UTR H in HEK293T cells transfected with scramble or Eif4a1 siRNA (right panel). The error bars represent the standard errors of the means (SEMs). ns, not significant; *p < 0.05, **p < 0.01 and ***p < 0.001. The data shown are representative of three independent experiments

We further examined this in a reporter assay. The Gins4 and Atcb 5’UTRs were cloned into the psiCheck2 reporter plasmid and placed in the 5’UTR of the humanized Renilla luciferase (hRLuc) gene (Fig. 7H). As shown in Fig. 7I, siRNA-mediated knockdown of eIF4A1 significantly downregulated the hRluc activity of the reporter gene containing the Gins4 5’UTR. Furthermore, CRISPR/Cas9-mediated deletion of Gins4 and Eif4a1 caused a similar impairment in B cell proliferation (Fig. 8A, B), suggesting that Gins4 is a major mediator of eIF4A1 control of cell cycle.

Fig. 8

eIF4A1 exerts its functions in B cells through Gins4 and other genes. A Immunoblot analysis of Gins4 and eIF4A1 protein expression in LPS-stimulated Cas9-GFP B cells transduced with retroviruses encoding nontargeting control (NTC), Eif4a1 or Gins4 sgRNA. The retroviral vector encodes blue fluorescent protein (BFP), and transduced cells are BFP positive. B Cas9-GFP and wild-type B cells (CD45.1 + ) were mixed at a 1:1 ratio, stimulated with LPS for 24 hours, transduced with retroviruses encoding nontargeting control (NTC), Eif4a1 or Gins4 sgRNA, and analyzed by flow cytometry at 72 hours post retroviral transduction. The proliferation ratio was calculated as indicated. C Scheme of iGCB culture. D Immunoblot analysis of eIF4A1, eIF4A2 and Gins4 proteins in Eif4a1fl/fl and Eif4a1fl/fl;Cγ1Cre B cells cultured in the iGCB system at the indicated time points. E, F Numbers of Eif4a1fl/fl and Eif4a1fl/fl;Cγ1Cre iGCB E and iPC F cells at the end of the indicated stages of culture. G Eif4a1fl/fl and Eif4a1fl/fl;Cγ1Cre B cells were cultured in the iGCB system and transduced with retroviruses encoding Eif4a1 or Gins4 at iGCB day 2.5. Transduced cells (GFP + ) were sorted at iGCB day 4 and replated on 40LB cells in the presence of IL-21 for iPC differentiation. The numbers of cells at the end of iPC culture were plotted. The data shown are representative of three independent experiments

We then asked whether ectopic expression of Gins4 is able to restore cellular proliferation of Eif4a1-deficient B cells. As Eif4a1fl/fl;CD19Cre B cells show impaired cellular proliferation and cannot be transduced by retroviruses, we bred Eif4a1fl/fl mice with Cγ1Cre mice, in which the expression of Cre is induced by transcription of the Igγ constant region gene segment (Cγ1) [33]. B cells from Eif4a1fl/fl;Cγ1Cre and Eif4a1fl/fl mice were analyzed in an in vitro culture system mimicking B-cell differentiation into plasma cells. In this system, naïve B cells were cultured on top of BALB/c 3T3 cells stably expressing CD40L and Baff (termed 40LB cells). In the presence of IL-4, naïve B cells acquire a GCB cell phenotype (Fas+GL-7+Bcl6+, termed iGCB cells) after 4 days of culture. Those iGCB cells can further differentiate into plasma cells (termed iPC cells) after 4 additional days of culture in 40LB cells in the presence of IL-21 (Fig. 8C) [34]. Eif4a1fl/fl;Cγ1Cre B cells showed significant reduction in eIF4A1 protein expression on day 2 and complete depletion on day 3 of the iGCB stage of culture (Fig. 8D). Consistent with previous observations in LPS-stimulated B cells (Fig. 7E), Eif4a1-deficient B cells expressed much lower amounts of the Gins4 protein than Eif4a1fl/fl B cells (Fig. 8D). The numbers of Eif4a1-deficient B cells at the end of the iGCB and iPC stages of culture were also much reduced (Fig. 8E, F). We then transduced Eif4a1fl/fl;Cγ1Cre B cells with retroviruses encoding eIF4A1 or Gins4 on day 2.5 of the iGCB stage. As shown in Fig. 8G, retroviral expression of eIF4A1 fully restored the number of Eif4a1fl/fl;Cγ1Cre B cells at the end of iPC culture, while retroviral expression of Gins4 had no obvious effect. Therefore, eIF4A1 likely controls cellular proliferation through multiple target genes, with Gins4 as one of them.