Fig 2: Mutually exclusive interaction of Nufip2 with Fmrp or Roquin. a Representative coomassie-stained PAGE of purified mFmrp (aa 1–132). b Biacore sensogram recording of the binding of NUFIP2 (aa 255–441) injected at 4 μM to surface-coupled Roquin-1 (aa 2–441) in the presence of increasing amounts (0.016–1 μM) of Fmrp (aa 1–132). The quality of Biacore experiments was assessed by two independent injections of 1 µm Fmrp and 4 µm NUFIP2 at different time points during each experiment. c Immunoprecipitation of endogenous NUFIP2 from lysates of HEK293T cells that were transiently transfected with Roquin-1 with or without co-transfection of FLAG-Fmrp using a monoclonal Nufip2 antibody. Immunoblot analysis to detect Nufip2, Fmrp and Roquin-1/2 after Nufip2 immunoprecipitation (IP) is shown. d–g Analysis of splenic CD4+ T cells from wild-type (wt) and Fmrp-deficient (Fmr1−/−) mice. d, e Flow cytometry analysis of CD4+ T cells showing naive (CD62Lhi CD44lo), effector-memory (CD62Llo CD44hi), or central-memory (CD62Lhi CD44hi) (d) or e Foxp3+ Tregs among CD4+ pre-gated T cells. f Flow cytometry analysis of ICOS expression on the surface of Foxp3+ splenic CD4+ T cells. g Quantified ICOS expression on CD4+ T cells isolated from Fmrp-deficient or wild-type mice in a 6-day in vitro culture. One representative of two (c) or three (b) independent experiments are shown in b and c. In d–f data are representative of four mice per genotype and g shows mean and SD of four mice per genotype

Fig 3: Deconvolution of siRNA pools to validate genuine hits. a Flow cytometry analysis of ICOS and mCherry expression in HeLa reporter cells treated with individual or pooled CNOT1 or NUFIP2 siRNAs (red) or non-targeting control siRNAs (siCtrl) (black). Prior to analysis, cells were treated with doxycycline for 18 h to induce Roquin-1 overexpression. b Quantified ICOS MFI of cells shown in a. Expression levels were normalized to siCtrl-treated cells. c qPCR analysis of CNOT1 or NUFIP2 mRNA expression in cells from a. Expression was calculated relative to the reference gene YWHAZ and normalized to siCtrl-treated cells. Error bars in b and c represent mean and SD of three independent experiments. In a representatives of three independent experiments are shown. Statistical significance in b was calculated with one-way ANOVA Kruskal−Wallis test followed by Dunn’s multiple comparisons test (*p < 0.05, **p < 0.01)

Fig 4: Direct physical interaction between Nufip2 and Roquin. Immunoblots were analyzed with antibodies against Nufip2 and Roquin, Roquin and GFP or Nufip2 and GFP. Anti-GFP or anti-Roquin immunoprecipitation from lysates of HEK293T cells transfected with the indicated expression vectors (a, b, e) and anti-Roquin (c) or anti-Nufip2 (d) immunoprecipitation of endogenous proteins from MEF cells of the indicated genotypes are shown. a Extracts were treated during immunoprecipitation with or without RNase as indicated and degradation of rRNA by RNase treatment was confirmed by ethidium bromide (EtBr) staining of RNA extracts from IP supernatants. f Representative coomassie-stained PAGE of purified Roquin-1 (aa 2–441) and NUFIP2 (aa 255–411) proteins. g, h Surface plasmon resonance study of the binding of NUFIP2 (aa 255–411) to immobilized Roquin-1 (aa 2–441). g Biacore sensogram recording the binding of NUFIP2 (aa 255–441) injected at two-fold serial dilutions ranging from 0.063 to 4 μM to Roquin-1 (aa 2–441). After the highest concentration, the 0.5 μM sample dilution was re-injected to assess potential accumulation of unspecific background binding. h Steady-state affinity analysis of the binding level in response units (RUs) against the Nufip2 (aa 255–411) protein concentration shown in a representative fitting curve. In a–e and g–h one representative of two (d) or three (a–c, e, g–h) independent experiments is shown. The KD in h was calculated from mean and SD of three independent experiments

Fig 5: Nufip2 protein is stabilized by Roquin. a Immunoblot analysis of Roquin, Nufip2, and Fmrp proteins in MEF lysates and supernatants or precipitates of b-isox-treated extracts. b Flow cytometry imaging analysis of GFP-Nufip2 localization in activated Th1 cells from Rc3h1fl/fl; Rc3h2fl/fl; Cd4-Cre-ERT2; rtTA mice. Before analysis, retrovirally transduced cells were treated with doxycycline for 24 h to induce GFP-NUFIP2 expression. c–f Immunoblot analysis of Nufip2 (c) or Nufip2 and Roquin (d–f) in c different mouse tissues, d mouse CD4+ T cells left untreated or stimulated for 12 h with (α-CD3) alone or in combination with anti-CD28 (α-CD28) or anti-ICOS (α-ICOS), e mouse CD4+ T cells with (Rc3h1/2fl/fl) or without (Rc3h1/2fl/fl; Cd4-Cre) Roquin expression, or f in lysates from wild-type and Roquin–deficient MEF cells, reconstituted with wild-type or Roquin-1 mutants as indicated. Gapdh (c–e) or α-tubulin (f) served as loading controls. g qPCR analysis of Nufip2 mRNA expression in cells from f. Expression was calculated relative to the reference gene Ywhaz and normalized to untransduced cells. Error bars in g represent mean and SD of two independent experiments. In a, b, d, e and f representatives of two (d, e, f) or three (a, b) independent experiments are shown