NSrp70 is a lymphocyte-essential splicing factor that controls thymocyte development

Abstract

Alternative pre-mRNA splicing is a critical step to generate multiple transcripts, thereby dramatically enlarging the proteomic diversity. Thus, a common feature of most alternative splicing factor knockout models is lethality. However, little is known about lineage-specific alternative splicing regulators in a physiological setting. Here, we report that NSrp70 is selectively expressed in developing thymocytes, highest at the double-positive (DP) stage. Global splicing and transcriptional profiling revealed that NSrp70 regulates the cell cycle and survival of thymocytes by controlling the alternative processing of various RNA splicing factors, including the oncogenic splicing factor SRSF1. A conditional-knockout of Nsrp1 (NSrp70-cKO) using CD4Cre developed severe defects in T cell maturation to single-positive thymocytes, due to insufficient T cell receptor (TCR) signaling and uncontrolled cell growth and death. Mice displayed severe peripheral lymphopenia and could not optimally control tumor growth. This study establishes a model to address the function of lymphoid-lineage-specific alternative splicing factor NSrp70 in a thymic T cell developmental pathway.

Figure 1.

NSrp70 is preferentially expressed in early embryonic tissues and in lymphoid cells. (A) Immunohistochemistry for NSrp70. Oocyte (top), morula (middle), and fetal embryo E11.5 (bottom) were isolated from WT mice, stained with anti-NSrp70 antibody followed by FITC-conjugated 2^nd antibody (green), TRITC-phalloidin (red), and DAPI (magenta and blue), and then visualized by confocal microscopy. (B) Fetal embryos from gestation day 11.5, 13.5, 15.5 and 17.5 mice were isolated. Nsrp1 mRNA was detected by RT-PCR (blots) and density was represented by bar graph. mGapdh was used as a loading control. E, embryonic day; bp, base-pair. (C) Intercrossing the heterozygous Nsrp1^+/GT mouse produced no offspring homozygous for the allele containing the gene-trap vector. GT, gene-trap vector; +/+, wild-type; +/GT, heterozygous Nsrp1^+/GT; GT/GT, homozygous Nsrp1^GT/GT. (D) Tissue distribution of NSrp70 was determined by western blot analysis in 8-week old mice. M.L.N, mesenchymal lymph node; L.N., lymph node; S.I., small intestine; L.I., large intestine; M, molecular mass (KDa). (E) Western blot (left) and RT-PCR (right) for NSrp70 in mouse immune cells. β-actin and mGapdh were shown as loading controls. SP, splenocytes. All data shown are representative of three independent experiments. The bar graphs indicate the mean ± standard deviation of the indicated protein blot or RNA gel densitometry presented with respect to β-actin or mGapdh (B and E).

Figure 2.

Development of CD4⁺ and CD8⁺ SP thymocytes is impaired in Nsrp1^f/fCD4Cre mice. (A) Western blot of NSrp70 expression in thymocyte subsets from Nsrp1^f/f (WT) and Nsrp1^f/fCD4Cre (cKO) mice. DN, double negative; DP, double positive; SP, single positive; WT, Nsrp1^f/f mouse; KO, Nsrp1^f/fCD4Cre mouse. The bar graphs indicate the mean ± standard deviation (SD) of the indicated protein blot densitometry presented with respect to β-actin. (B and C) Staining for CD4 (green) and CD8 (red) in thymus (B) and in spleen and lymph node (C) from 6-week-old WT and KO mice. White arrowheads: CD8⁺ SP thymocytes in medulla. M, medulla; C, cortex. Original magnification, ×10. The fluorescent intensity of CD4⁺ and CD8⁺ thymocytes was quantified (C, bottom). (D) Flow cytometric analysis of CD4 and CD8 on thymocytes. (E) Quantification of average percent (top) and cell numbers (bottom) of DN, DP, CD4⁺ or CD8⁺ SP thymocyte subpopulations. Each black and white circle in graphs of cell numbers represents an individual mouse. The bar graphs (top) and small horizontal lines (bottom) indicate the mean ± SD. *, meaningful P-value; NS, non-significant P-value. (F) Surface staining of TCRβ and CD69 on total thymocytes. (G–I) Quantification of TCRβ^hi thymocytes population gated on III and IV (G), total cell numbers gated on I–IV gate plots (H), and CD4⁺CD69^hi and CD8⁺CD69^hi cells (I) from panel (F). The bar graphs indicate the mean ± SD of populations. hi, high expression. Each black and white circle in graphs represents an individual mouse. The small horizontal lines indicate the mean ± SD. All data shown are representative of three independent experiments.

Figure 3.

Global alternative RNA splicing changes in NSrp70-cKO DP thymocytes. (A) The number of isoforms and genes present in the given samples. We checked the number of isoforms and genes present in given wild-type and KO samples and found significant reductions of the numbers of isoforms and genes in DP thymocytes (6.13% and 5.5% reductions, respectively; Wilcoxon rank sum tests, one-sided, P-values ≤ 0.05). (B) Venn diagram of the number of significantly alternative spliced-genes from three mRNA-seq datasets of Nsrp1^f/f (WT) and Nsrp1^f/fCD4Cre (KO) DP thymocytes. PSI, Percent spliced in; BF, Bayes’ factor. (C) Quantification of the different alternative splicing events affected by NSrp70. (D) The relative percent of each alternative splicing event affected by NSrp70. The small horizontal lines indicate the mean ± standard deviation *, meaningful P-value. (E, F) Gene ontology analysis (E) and functional association network (STRING) (F) of alternative spliced-targets affected by NSrp70-cKO. Fisher exact P-values were plotted for each enriched functional category. (G) Sashimi plot of alternative exons affected by NSrp70. Genes were chosen to represent both an increase and a decrease of PSI, and the numbers of exon junction reads are indicated. (H) PSI and BF values of different types of NSrp70-regulated alternative splicing events based on MISO analysis.

Figure 4.

NSrp70 controls the expression of SRSF1 and counteracts mutually. (A) Immunoprecipitation (left) and immunofluorescence images (right) of NSrp70 and SRSF1. HEK293T cells were transfected with GFP (empty vector), GFP_NSrp70 (G_NSrp70), or RS1M mutant. In some cases, mCherry-fused SRSF1 and HDAC1 were co-transfected. Samples were immunoprecipitated and blotted with antibodies against the indicated proteins (left). IP, immunoprecipitation; WB, western blotting; M, molecular mass (kDa). Fluorescence signals were visualized under a confocal microscope (right). Magnification, 100×. Results are representative of three independent experiments. (B) RIP assay for validation of NSrp70-binding mRNA. The samples in (A) were immunoprecipitated with the indicated antibodies (anti-rabbit IgG or anti-GFP). RNAs purified from the IP samples and total lysates were determined by RT-PCR. bp, base pair. (C) Schematic diagram of wild-type NSrp70 and RS1M mutant. Potential regulation of alternative splicing and speckle organization by NSrp70 and its effect on T cell development. NLS, nuclear localization sequence. (D) RT-PCR analysis of different SRSF1 isoforms in Nsrp1^f/f (WT) and Nsrp1^f/fCD4Cre (KO) DP thymocyte. (E) Real-time quantitative PCR (left) and western blot analysis (right) for SRSF1 and HDAC1 from WT and KO DP thymocytes. Gapdh and β-actin were shown as the loading controls. *, meaningful P-value. (F) NSrp70 counteracts SRSF1 in vivo splicing assay. HEK293T cells were co-transfected with the indicated constructs and Fas minigene. Exon inclusion or exclusion was determined by RT-PCR (top). The ratio of exclusion or inclusion of Fas exon 6 is shown as the normalized ratio (%) (middle). mCherry, empty vector; mC_SRSF1, mCherry_SRSF1. (G) NSrp70 effects on the proliferation of EO771 cancer cells. The cells were stably transfected with NSrp70, SRSF1, or a control vector and analyzed by colony formation assays with mean ± standard deviation of relative colony numbers plotted (left). Expression of NSrp70 and SRSF1 were confirmed (right). All data shown are representative of three independent experiments. *, meaningful P-value.

Figure 5.

Aberrant gene expression related to the cell cycle and TCR intracellular signal transduction in NSrp70-cKO DP thymocytes. (A) Hierarchical clustering of gene expressions with more than two-fold changes. We found distinct gene clusters of up- or down-regulation in Nsrp70-cKO DP thymocytes. The heat-map for gene expression patterns was generated using the Multi Experiment Viewer (MeV) software. Genes with red and blue colors indicate higher and lower expressions, respectively. fc2, 2-fold change. (B) Top-5 most significantly up-regulated or down-regulated biological process terms from significantly differentially expressed genes (negative binomial tests) based on gene ontology enrichment tests (modified Fisher's exact tests from DAVID). (C) The global map of significantly up-regulated (pink) or down-regulated (skyblue) gene ontology terms (biological processes and subcellular locations, top and bottom, respectively) by gene ontology enrichment tests (P-value < 0.01). Like in the Enrichment Map (54), we clustered enriched biological processes and subcellular locations as connected in the map if they shared genes in significant numbers (Jaccard > 0.25). (D) Real-time quantitative PCR analysis for the validation of NSrp70-regulated targets. mGapdh was used as the loading control. *, meaningful P-value. All data shown are representative of three independent experiments.

Figure 6.

Deletion of NSrp70 induces uncontrolled cell proliferation followed by apoptotic cell death. (A) Cell cycle analysis of DP thymocytes from Nsrp1^f/f (WT) and Nsrp1^f/fCD4Cre (KO) mice. Populations of CD69^– or CD69⁺ thymocytes were gated from CD4 and CD8 DP thymocytes (left) and were analyzed for DNA content by 7-ADD intensity (right). The bar graphs indicate average ± SD of different cell cycle stages population. *, meaningful P-value. (B, C) Analysis of cell proliferation by Ki-67 staining and apoptotic cell death by annexin V and 7-ADD. DP thymocytes from (A) were stained with anti-Ki-67 antibody and 7-ADD (B) or annexin V and 7-ADD (C) and analyzed by flow cytometry. Bar graphs indicate mean fluorescence intensities (MFI) (B, bottom). Annexin V⁺ populations represent early apoptotic cells and annexin V⁺ and 7-ADD⁺ populations represent dead cells (C). The bar graphs indicate average ± SD of apoptotic and dead thymocyte populations. NS, non-significant P-value. (D) Western blot of cell cycle and cell death-related proteins in samples extracted from WT and KO thymocytes. β-actin served as a loading control. The bar graphs indicate average ± SD of indicated protein blot densitometry presented relative to β-actin. All data shown are representative of three independent experiments.

Figure 7.

Deletion of NSrp70 results in defective survival signals following TCR activation in CD69⁺ DP thymocytes. (A) KEGG T cell receptor signaling pathway. The blue asterisks (*) represent down-regulated genes from the RNA-seq analysis (Figure 5B). (B) Expression of TCRβ, CD3ϵ, and CD3ζ on CD69⁺ DP thymocytes from Nsrp1^f/f (WT) and Nsrp1^f/fCD4Cre (KO) mice. (C) A schematic model of gene regulation by NSrp70. NSrp70 sequesters splicing factors in the nuclear speckles. Disintegration of splicing factors by NSrp70 deletion induces abnormal gene regulation during thymocyte development. As one of the results, reduced TCR expression may cause impaired T cell maturation. (D) Calcium flux in DP thymocytes. Cells from (A) were stimulated with PMA and ionomycin (P/I) or anti-CD3/CD28 antibodies, and then calcium fluxes were measured by flow cytometry. (E) Western blot of ZAP70, PKCθ, Erk1/2, and p38 in DP cell lysates stimulated on anti-CD3/28 for 0, 5, and 20 min. β-actin served as the loading control. M, molecular mass (KDa). (F) In vitro thymocyte development assay. CD69^– DP thymocytes were stimulated on anti-TCRβ/CD2 antibodies for 20 h (stimulation), or the cells were further incubated for 20 h in medium without stimulation (recovery). (G and H) Cells from (F) were stained for Ki-67 (G) or annexin V and 7ADD (H). Cells were analyzed by flow cytometry (F–H). The bar graphs indicate mean fluorescence intensities (MFI) (G). *, meaningful P-value; NC, non-coated; S, stimulation; R, recovery. The bar graphs indicate average ± standard deviation of apoptotic and dead thymocytes population (H). NS, non-significant P-value. All data shown are representative of three independent experiments.

Figure 8.

NSrp70 deficiency results in severe peripheral lymphopenia. (A, B) Flow cytometric analysis of CD4 and CD8 on T cells from lymph node (top) and spleen (bottom) of WT and KO mice. Quantification of CD4⁺ and CD8⁺ populations (A) and cell numbers (B) were represented as bar graphs (A) and circle symbols (B). Small horizontal lines indicate the mean ± standard deviation. Each black and white circle represents an individual mouse. *, meaningful P-value. (C) Analysis of CD62L and CD44 on T cells from (A). Numbers in quadrants indicate percent cells in each throughout. (D) Western blot analysis of NSrp70 in CD3⁺ T cells (left) and anti-CD3/28 and IL-2-induced CD3⁺ T blasts (right) obtained from WT and KO lymph nodes. M: molecular mass (KDa). β-actin served as a loading control. (E and F) Flow cytometric analysis of CD4⁺ and CD8⁺ and Ki-67⁺ and 7-ADD⁺ (E) and TCRβ, CD28, and CD25 (F) on CD3⁺ T blasts from (D). Numbers in the areas indicate percent cells in each throughout. (G) Calcium flux of CD3⁺ T blasts from (D). Calcium flux was measured as described in Figure 6D. (H) Schematic experimental design of B16F10 tumor model. (I) B16F10 cells were subcutaneously injected into left and right flank of WT and KO mice. Mice were sacrificed after 2 weeks. Tumor sizes and weights were determined. All data shown are representative of three independent experiments. *, meaningful P-value.