Fig 2: TFIP11 knockdown induces intron retention.a Number of differential alternative 5' splice site (A5SS), alternative 3' splice site (A3SS), mutually exclusive exon (MXE), retained intron (RI), and skipped exon (SE) events identified with rMATS in cells transfected for 72 h with one TFIP11 siRNA (siTFIP11 #1) compared to control siRNA (siCtr). b–g RT-qPCR shows increased intron retention in mRNA encoding NXF1 (DPSI by RNA-seq: 0.07; overall p = 8.4e-4, p = 0.0027 for siTFIP11#1, p = 0.0032 for siTFIP11#2), THOC6 (DPSI by RNA-seq: 0.14; overall p = 0.0015, p = 0.0049 for siTFIP11#1, p = 0.042 for siTFIP11#2), SMC4 (DPSI by RNA-seq: 0.01, overall p = 0.0017, p = 0.0054 for siTFIP11#1, p = 0.0041 for siTFIP11#2), sororin (DPSI by RNA-seq: 0.11; overall p = 0.0041, p = 0.0072 for siTFIP11#1, p = 0.0052 for siTFIP11#2), CENPE (DPSI by RNA-seq: 0.09; overall p = 3.79e-4, p = 0,001 for siTFIP11#1, p = 0.0086 for siTFIP11#2) and Alyref (DPSI by RNA-seq: 0.06; overall p = 0.0014, p = 0.0055 for siTFIP11#1, p = 0.0064 for siTFIP11#2) in cells transfected with one of two TFIP11 siRNAs (siTFIP11 #1 and siTFIP11 #2) compared to control siRNA (siCtr) for 72 h or mock-transfected cells (No siRNA). n = 3 independent experiments. p-values calculated using one-way ANOVA with Tukey post-hoc test. Only p-values < 0.05 (relative to siCtr) are displayed. Box limits = 25th to 75th percentiles; line = median; whiskers = min to max. h Western blotting in HeLa cells transfected as in b shows decreased abundance of indicated proteins whose mRNAs have increased IR. i Sequences at 5' and 3' splice sites (SS), branch points and polypyrimidine tracts (PPT) for introns with increased retention in cells transfected as in a (n = 2311 introns) and introns from all detected splicing events (n = 104,230 introns). j Length of introns and their flanking exons. Control and retained introns as in i. p-values calculated using Welch’s two-sample t-test. k GC content near 5' and 3' splice sites. Control and retained introns as in i. l–m Strength and length of PPT of introns. Control and retained introns as in i. p-values calculated using Welch’s two-sample t-test. n Strength of 5' and 3' splice sites (SS). Control and retained introns as in i. MaxEntScores = 0 are displayed. p-values calculated using all values with Welch’s two-sample t-test. Source data are provided as a Source Data file.

Fig 3: TFIP11 localizes in nuclear speckles, Cajal Bodies (CBs) and nucleoli.a Immunoprecipitation (IP) of endogenous TFIP11 from HeLa cells followed by western blotting (WB) for indicated proteins. b Representative image of endogenous TFIP11 and DHX15 interaction (in red) assessed by in situ proximity ligation assay (iPLA). Nucleus was counterstained in blue. Scale bar = 5 µm. c Quantification of TFIP11/DHX15 interaction detected by iPLA in HeLa cells transfected for 48 h with control siRNA (siCtr n = 293) or DHX15 siRNA (siDHX15 #1 n = 38). Box limits = 25th to 75th percentiles; line = median; whiskers = min to max. p-value calculated using unpaired two-tailed t-test (p < 0.0001). d HeLa cells co-stained with anti-DHX15 (in green) and anti-TFIP11 (in red) antibodies. Nucleus was counterstained in blue. Individual channels, merged channels and magnification of boxed regions are shown. Scale bar = 10 µm. e–n HeLa cells co-stained for TFIP11 (in green) and SC35 (e), coilin (f), SMN (g), FBL (h), or NOP58 (i) (in red). Individual channels, merged channels with nuclear staining (in blue) and magnification of boxed regions are shown. Scale bar = 5 µm. White arrows indicate diffuse nucleolar staining for TFIP11 with occasional more intense foci. j–n Intensity plots for regions in e–i (white arrows) indicate co-localization of TFIP11 with each corresponding protein (black arrows). Blue dashed lines indicate nucleus edges. o–p Endogenous TFIP11 in U2OS cells detected by immunogold/electron microscopy (EM). White arrows in (o) show 6-nm gold particles in white circles, indicating TFIP11 in perichromatin fibrils (PF) at the periphery of interchromatin granule clusters (IGC, white dashed line). Scale bar = 0.25 µm. White arrows in p indicate particles labeling TFIP11 in nucleolar compartments: dense fibrillar center (DFC), fibrillar center (FC) and granular component (GC). TFIP11 is also at the periphery of the nucleolus, delimited by the white dashed line. Scale bar = 0.25 µm. q Quantification of immunogold-labeled TFIP11 in the DFC, FC and GC in U2OS cells. Negative control = no primary antibody. N = 15 different nucleolar fields from 15 different cells. p-value calculated using two-tailed paired t-test. GC p < 0.0001; DFC p = 0.0035; FC p = 0.0107. Box limits = 25th to 75th percentiles; line = median; whiskers = min to max. Source data are provided as a Source Data file.

Fig 4: TFIP11 is required for U4/U6.U5 tri-snRNP stability.a Immunoprecipitation (IP) of endogenous Sm proteins from HeLa cell extracts pre-treated or not with RNAse A followed by western blotting (WB) for the indicated proteins. b Immunoprecipitation (IP) of endogenous TFIP11 from HeLa cell extracts followed by western blotting (WB) for the indicated snRNP-specific proteins. c Immunoprecipitation (IP) of endogenous Sm protein from extracts of HeLa cells mock-transfected (No siRNA) or transfected for 48 h with one TFIP11 siRNA (siTFIP11 #1) or control siRNA (siCtr) followed by western blotting (WB) of the indicated snRNP-specific proteins. d, e Glycerol-gradient fractionation. d Northern blotting (NB) analysis of U1, U2, U4, U5 and U6 snRNAs isolated from a glycerol-gradient (10–30%) fractionation of nuclear extracts from siCtr- and siTFIP11 #1-transfected cells 72 h post-transfection. NB data shown are representative of two independent experiments. e Western blotting (WB) analysis of indicated proteins isolated from the same glycerol-gradient (10–30%) fractions. f Densitometry scanning of NB autoradiographs for each U snRNA in each fraction of nuclear extracts from siCtr- and siTFIP11 #1-transfected cells. g Western blotting for the indicated proteins on the nuclear extracts (NE) of HeLa cells transfected for 72 h with control siRNA (siCtr) or with TFIP11 siRNA (siTFIP11 #1). h, i HeLa cells were mock-transfected (No siRNA) or transfected for 48 h with one of two different TFIP11 siRNAs (siTFIP11 #1 and siTFIP11 #2) or control siRNA (siCtr), then co-stained with anti-coilin antibody (in red) and antibodies against Prp4K (h) or EFTUD2 (i) (in green). Merged channels with nuclear staining (in blue) and magnification of boxed regions are shown. Scale bar = 5 µm. Source data are provided as a Source Data file.

Fig 5: TFIP11 knockdown alters morphology of Cajal Bodies (CBs), nuclear speckles and nucleoli.a HeLa cells were mock-transfected (No siRNA) or transfected for 48 h with one of two different TFIP11 siRNAs (siTFIP11 #1 and siTFIP11 #2) or control siRNA (siCtr). Cells were co-stained with anti-coilin (in red) and anti-NOP58 (in green) antibodies. Individual channels and merged channel with nuclear staining (in blue) and magnification of boxed regions are shown. Scale bar = 5 µm. b Quantification of the number of Cajal Bodies (CBs) in HeLa cells transfected as in a (No siRNA n = 363/siTFIP11 #1 n = 247/siTFIP11 #2 n = 297/siCtr n = 289 from four independent experiments). c Quantification of the number of cells with coilin foci in the nucleolus (No siRNA n = 343/siTFIP11 #1 n = 540/siTFIP11 #2 n = 634/siCtr n = 431 from three independent experiments). p-values for panels b–c were calculated by Kruskal–Wallis test followed by Dunn post-hoc test with Benjamini–Hochberg correction. Adjusted p-values < 0.05 (relative to siCtr) are displayed. Box limits = min to max; line = median. d HeLa cells transfected as in a and co-stained with anti-coilin (in red) and anti-SC35 (in green) antibodies. Individual channels and merged channel with nuclear staining (in blue) and magnification of boxed regions are shown. Scale bar = 5 µm. e, f Transmission electron microscopy (TEM) showing Cajal Bodies (CB) of HeLa cells transfected for 48 h with control siRNA (siCtr) or TFIP11 siRNA (siTFIP11 #1). IGC = interchromatin granule clusters. Nu = nucleolus. NB = nuclear body. Scale bar = 0.25 µm. g TEM showing interchromatin granule clusters (IGC) of HeLa cells transfected as in e. Scale bar = 0.2 µm. h TEM showing nucleoli of HeLa cells transfected as in e. The three classical compartments (the dense fibrillar center–DFC, the fibrillar center–FC, and the granular component–GC) of the nucleolus (delimited by white dashed line) are indicated. NuB = Nucleolar Body. Scale bar = 0.2 µm. Source data are provided as a Source Data file.