Fig 2: SRSF1 is required for thymocyte proliferation and survival.(A and B) Apoptosis in postselection thymocyte subsets was analyzed with annexin V staining. Representative histograms (A) show the percentages of annexin V+ cells in indicated subsets from control (n = 6) and Srsf1fl/flLckCre/+ (n = 7) mice. Cumulative data on frequency of annexin V+ cells are shown in (B). (C and D) BrdU incorporation was detected in TCRβloCD69−CD4+CD8+ DP cells (pre-DP), TCRβhiCD69+CD24+CD4+CD8+ DP cells (post-DP), mature TCRβhiCD69−CD24−CD4+ SP (M-CD4), and mature TCRβhiCD69−CD24−CD8+ SP (M-CD8) thymocytes, respectively. Representative contour plots (C) show BrdU+ cells in indicated subsets from control (top row) and Srsf1fl/flLckCre/+ mice (bottom row). Cumulative data on frequency of BrdU+ cells are shown in (D) (n = 7 per group). (E and F) IL-7Rα expression on mature TCRβhiCD69−CD24−CD4+ and TCRβhiCD69−CD24−CD8+ SP thymocytes was detected from control and Srsf1fl/flLckCre/+ mice (n = 6 per group) (E). Cumulative data on geometric mean fluorescence intensity (gMFI) are shown in (F). The geometric mean fluorescence intensity of IL-7Rα from control cells was set as 1, and its relative expression in cells from Srsf1fl/flLckCre/+ mice was normalized accordingly. (G) qPCR validation of genes involved in apoptosis in mature CD4+ and CD8+ SP thymocytes from control (n = 5) and Srsf1fl/flLckCre/+ (n = 4) mice. The relative expression of each transcript (after normalization to Gapdh) in control cells was set as 1 and that in cells from Srsf1fl/flLckCre/+ mice was normalized accordingly. Cumulative data are means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 (Student’s t test). All data are from at least three independent experiments.

Fig 3: SRSF1 regulates T cell development in a cell-intrinsic manner.(A) Flowchart of generation and analysis of BM chimeric mice. (B) Representative pseudocolor plot show the classic subsets of thymocytes with CD4 and CD8 staining from the chimeras generated with control and Srsf1fl/flLckCre/+ donor (CD45.2+) BM cells. (C) Frequency of indicated subsets as in (B) is shown accordingly (n = 6 per group). (D) Characterization of postselection thymocytes in chimeric mice. TCRβhi postselection thymocytes (CD45.2+) derived from control and Srsf1fl/flLckCre/+ mice in chimeras are shown by contour plot, and the gating strategies were consistent with those described in Fig. 1E. (E) Frequency of each subpopulation in (D) is statistically shown (n = 6 per group). Cumulative data are means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 (Student’s t test). Data are from at least three independent experiments.

Fig 4: SF2/ASF interacts with NS mRNA through multiple binding sites including the ESE motif and A540G substitution enhances SF2 binding to NEP ESE.(a) Schematic illustration of the SF2/ASF-binding sites in NS mRNA and locations of probes used in the following experiments. (b) SF2/ASF binds to NS (529–548), in which the putative ESE is located, in RNA EMSA using NE containing FLAG-tagged SF2/ASF. α-FLAG antibody was used for super-shifting and IgG was used as a control antibody. (c) Different regions of NS mRNA bound by SF2/ASF were analysed by RNA EMSA using FLAG-SF2ΔRS. RNA probes derived from the splicing donor site (DS), acceptor site (AS), exon-1 (37–56) and exon-2 (529–548) of NS mRNA were similarly tested, as in b. (d) SF2 binding efficiency to WT and A540G mutant ESE was compared in RNA EMSA with increasing amounts of FLAG-SF2 NE. (e) Competition efficiency with WT or A540G cold probes was compared in RNA EMSA with increasing amounts of cold probes (competitors). Results displayed in d,e are representative of three separate experiments. Error bars represent mean±s.d. (n=3). Statistical significance was analysed by Student's t-test: *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. (f) SF2 was purified by IP in high-salt RIPA buffer using α-FLAG antibodies and the purity of FLAG-SF2 verified by Coomassie blue staining. Efficiency of SF2 binding to WT and A540G mutant ESEs was compared in RNA EMSA with increasing amounts of purified FLAG-SF2. The relative intensity of the shifted bands was quantified using Image-J and is shown at the right side of each graph (d,e). (g) RNA-IP assay with lysates prepared from pHW2000-H7N9-NS transfected cells co-transfected with FLAG-SF2, FLAG-vector or mock transfected (NT). Whole-cell lysates of HEK293T cells were prepared at 48 h post transfection and subjected to IP with α-FLAG. (h) RNA-IP assay in virus infected cells. Whole-cell lysates were prepared from A549 cells infected with rH9N2-WT virus (multiplicity of infection (MOI)=2) at 16 h post infection and subjected to IP with α-SF2/ASF (endogenous). (g,h) RNA-IPs were followed by RNA extraction and RT–qPCR with primers detecting viral NS, cellular U87 scaRNA or viral PB1 mRNA. Fold enrichment of mRNA was calculated by the ΔCt method. The error bars represent mean±s.d. (n=3).

Fig 5: SRSF1, SRSF3, hnRNPK, hnRNPM, and FUS bind DHX9 exon 6A. (A) In silico analysis was performed using the Splice Aid database to identify putative consensus motifs for RBPs in the exon 6A sequence. In (A), schematic representation of DHX9 alternative exon 6A, with the position of SRSF1 (blue), SRSF3 (red), hnRNPK (green), hnRNPM (grey), and FUS (yellow) binding sites. Arrows indicate the primers’ positions used for the amplicons along the sequence. (B) TC-71 cells were UV-crosslinked, and protein-RNA extracts were immunoprecipitated with either control IgGs or hnRNPM, hnRNPK, FUS, SRSF1, and SRSF3 antibodies. Experiments were performed at least three times. Histograms show RT-qPCR analysis of the cross-linked and immunoprecipitation (CLIP) assay to analyze the binding of the indicated RNA binding proteins (RBPs) to intron 6 and exon 6A of DHX9 pre-mRNA. Results are expressed as a percentage of input (± S.D.). Statistical analysis was performed by one-way ANOVA with Bonferroni post-hoc test (p-value: *** < 0.001).