Fig 2: Colocalization of the components of Mediator, LEC, NELF and HLF in Cajal bodies.a–g HeLa cells were fixed by formaldehyde and stained with anti-ICE1 (magenta), anti-MED26 (magenta), anti-MED6 (magenta), anti-MED24 (magenta), anti-NELFb (magenta), anti-CSTF64 (magenta), anti-AFF4 (magenta), and anti-coilin (green) antibodies. Scale bars, 5 µm. h Quantification of the number and signal intensity of ICE1 particles in HeLa cells. i Quantification of the number and signal intensity of MED26 particles in HeLa cells.

Fig 3: LEC copurifies with transcription termination factors including CBCA-NELF-DSIF, HLF, and Integrator.a Schematic representation of the strategy for purifying LEC-binding proteins. Nuclear extract fractions from parental 293FRT cells and 293FRT cells stably expressing FLAG-tagged ICE1 were prepared in the presence of Benzonase® Nuclease. ICE1-binding proteins were purified from nuclear extracts through anti-FLAG affinity purification. b Silver staining of F-ICE1-binding proteins purified through anti-FLAG affinity chromatography. Each band of F-ICE1-binding proteins was cut out from the gels. Each gel was treated with trypsin and the resulting peptides from the gels were analyzed by mass spectrometry. c F-ICE1-binding proteins purified through anti-FLAG affinity chromatography or non-specific proteins present in negative FLAG control were identified through MudPIT (multidimensional protein identification technology)-based proteomic analysis. To estimate the relative protein levels, distributed normalized spectral abundance factors (dNSAF) were calculated for each detected protein57,62–64.

Fig 4: LEC, but not SEC, is required for efficient 3'-end processing of RDH genes.a Knockdown of MED26 and ICE1, but not AFF4, increased the ratio of unprocessed transcripts (UTs) to coding transcripts (CTs) of RDH genes, but not c-myc or GAPDH genes. Total RNAs were extracted from cells treated with control, MED26, ICE1, or AFF4 siRNAs, and unproccessed and coding regions were transfected and the ratio of UTs to CTs was measured by real-time qPCR. Data points are the mean of three independent experiments and error bars show the standard deviation. The P values for the indicated comparisons were determined by Student’s t test (*P < 0.05; **P < 0.01). n = 3 biologically independent samples. Source data are provided as a Source Data file. b Heat map showing differences in transcript abundance in HEK293T cells transfected with either control or ZC3H8 siRNAs (log2 siRNA ZC3H8/control), determined by RNA-seq of polyA-selected libraries. c ZC3H8 knockdown increased the ratio of UTs and CTs of RDH genes. Data points are the average of three independent experiments and error bars show standard deviation. The P values for the indicated comparisons were determined by Student’s t test (*P < 0.05; **P < 0.01). n = 3 biologically independent samples. Source data are provided as a Source Data file. d Venn diagram showing overlap of genes upregulated following knockdown of MED26, ICE1, and AFF4. The red box highlights a group of 124 genes increased by MED26 and ICE1 knockdown, but not by knockdown of AFF4. e The top 20 genes in groups affected by MED26 and ICE1 knockdown, but not by knockdown of AFF4, include many RDH genes. n.s., Not significant.

Fig 5: ICE1 is required for occupancy of the components of HLF and Integrator.a Knockdown of ICE1 decreased the occupancy of CPSF3 at RDH genes. Ct values of each ChIP were normalized to that of input. Each value is the average of three independent experiments and error bars show standard deviation. The P values for the indicated comparisons were determined by Student’s t test (*P < 0.05; **P < 0.01). n = 3 biologically independent samples. Source data are provided as a Source Data file. b Knockdown of ICE1 decreased the occupancy of INTS9 at snRNA genes. Ct values of each ChIP were normalized to that of the input. Each value is the average of three independent experiments and error bars show the standard deviation. The P values for the indicated comparisons were determined by Student’s t test (*P < 0.05; **P < 0.01). n = 3 biologically independent samples. Source data are provided as a Source Data file. c Knockdown of MED26 decreased the occupancy of CPSF3 at RDH genes. The Ct values of each ChIP were normalized by that of the input. Each value is the mean of three independent experiments and error bars show the standard deviation. The P values for the indicated comparisons were determined by Student’s t test (*P < 0.1; **P < 0.05). n = 3 biologically independent samples. Source data are provided as a Source Data file. d Knockdown of MED26 decreased the occupancy of INTS9 at snRNA genes. The Ct values of each ChIP were normalized by that of the input. Each value is the mean of three independent experiments and error bars show the standard deviation. The P values for the indicated comparisons were determined by Student’s t test (*P < 0.05; **P < 0.01). n = 3 biologically independent samples. Source data are provided as a Source Data file.