Fig 2: Expected pathway of calcium-mediated SMN splicing regulation. BFA, thapsigargin (TG) and calcium ionophore commonly increase the cytosolic calcium concentration, which is critical for the rescue of the SMN2 exon 7 splicing defect. In this pathway, CaMK4 may relay the stimulatory signal of the calcium to splicing factors, such as hnRNP M, PSF, Tra2-ß and hnRNP A1, and consequently promote exon 7 inclusion in SMN splicing (black fold in the bottom). Thin and short arrows (upward and downward) indicate the increase or the decrease of RNA-binding or protein amount of trans-acting factors. The numbers (6, 7, and 8) indicate exon 6, 7, and 8 of SMN2 pre-mRNA, respectively.

Fig 3: DAP3 facilitates the binding of splicing factors SFPQ and NONO to target RNAs.a Flowchart of the immunoprecipitation coupled to mass spectrometry (IP-MS) in combination with SILAC (stable isotope labelling with amino acids in cell culture) to detect DAP3 interactors. b Scatterplot of DAP3-immunoprecipitated proteins retrieved from both forward and reverse SILAC-based IP-MS. DAP3 and several top interactors (e.g., SFPQ and NONO) are highlighted. c Co-IP analysis of protein extracts from the WT and DAP3-KO EC109 cells. IP was performed with a DAP3 antibody, followed by western blot analysis of DAP3-pulldown products using DAP3, SFPQ, and NONO antibodies. d, e Co-IP analysis of protein extracts from EC109 cells. IP was performed with a SFPQ (d) or NONO (e) antibody, followed by western blot analysis of SFPQ or NONO-pulldown products using the indicated antibodies. IgG antibody was used as negative control. f Co-IP analysis of protein extracts from EC109 cells. RNase A treated (RNase A+) or untreated (RNase A-) protein lysates were used for IP using DAP3 antibody, followed by western blot analysis of DAP3-pulldown products using the indicated antibodies. IgG antibody was used as negative control. Agarose gel demonstrating the successful digestion of total RNA. g Semiquantitative RT-PCR analyses of the indicated splicing events upon overexpression of SFPQ or NONO alone or together in EC109 cells. h Semiquantitative RT-PCR analyses of the indicated splicing events upon overexpression of SFPQ or NONO in DAP3-KD EC109 cells. i RIP-qPCR analysis of the association between DAP3 protein and the indicated mRNA transcripts (WSB1, SSBP3, and RHBDD2). Data are represented as mean ± s.d. of n = 4 biologically independent samples. j RIP-qPCR analysis of the binding of SFPQ or NONO to the indicated mRNA transcripts in DAP3-KO and WT EC109 cells that were transfected with Flag-tagged SFPQ or NONO, respectively. RIP was conducted using anti-Flag M2 beads. g, h, j Data are represented as mean ± s.d. of n = 3 biologically independent samples. g–j Statistical significance is determined by unpaired, two-tailed Student’s t-test (*p < 0.05, **p < 0.01). Exact p-values and source data are provided in Source Data file.

Fig 4: Neat1 is released from nuclear paraspeckles into cytoplasm during inflammasome activation. a–c Cell lysates of untreated iBMDMs (a–c), LPS-primed iBMDMs (a–c), nigericin-activated iBMDMs (a), flagellin-activated iBMDMs (b), or poly(dA:dT)-activated iBMDMs (c) were analyzed by Western blotting. d Colocation of paraspeckle proteins, Sfpq (green), and Pspc1 (red) were assayed by immunostaining. The location of paraspeckles (yellow) was obtained by merging two signals of Sfpq and Pspc1. The position of the nucleus is shown in the bright-field image. e, f Interactions among paraspeckle components in untreated iBMDMs (e, f), LPS-primed iBMDMs (e, f), flagellin-activated iBMDMs (e), or poly(dA:dT)-activated iBMDMs (f) were analyzed by co-immunoprecipitation assay. g, h iBMDMs infected with lentiviruses expressing control, Pspc1, Sfpq, or Nono shRNA were analyzed for the cytoplasmic and nuclear levels of Neat1 and Neat1_2. Percentages of cytoplasmic Neat1 or Neat1_2 among total Neat1 or Neat1_2 RNA were shown in h. In d, the bar graph indicates 10 µm. In g and h, data shown are mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed t-test. Source data are provided as a Source Data file

Fig 5: Multilayered control of splicing regulatory networks by DAP3 leads to widespread alternative splicing changes in cancer.There are two distinct mechanisms of how DAP3 exerts its splicing regulatory functions. First, DAP3 directly binds to target RNA and mediates the recruitment of splicing factors, such as SFPQ and NONO, to the binding sites. Another mechanism involves indirect modulation of splicing through fine-tuning the splicing pattern of hundreds of splicing factors by DAP3. When DAP3 is overexpressed in many cancer types, global splicing changes can be observed and contribute to tumorigenesis.