Fig 2: Double SCAF4 and SCAF8 Knockout Is Lethal(A) Domain structure of SCAF4 and SCAF8.(B) Scheme for generation of single and double SCAF4 and SCAF8 CRISPR knockouts (KOs). 4KO, single SCAF4 KO; 8KO, single SCAF8 KO; dKO, double KO.(C) Dox-inducible GFP-SCAF expression in WT and KO cell lines. Left, western blot before and after Dox removal. Asterisk between lanes for day 2 and 3 indicates non-specific band. Right, quantification of relative GFP-rescue protein levels. Error bars represent ±SD.(D) Colony formation assays, with cells grown either with (left, +GFP-SCAF4; right, +GFP-SCAF8) or without (no rescue) Dox for 5 days prior to seeding single cells for colony formation.(E) Quantification of colony formation assays from two biological replicates (each seeded in triplicate). Error bars represent ±SD.(F) Cellular localization of GFP-SCAF4 or GFP-SCAF8 in HEK293 cells.See also Figure S1.

Fig 3: Identification of polyA Site Changes in SCAF4 and SCAF8 KOs, Related to Figure 5(A) Significant MISO events (bayes factor = 10 & dPSI = +/-0.3) identified in SCAF4 KO, SCAF8 KO and SCAF4 SCAF8 dKO compared to WT. Event types are abbreviated as: A3SS: Alternative 3' splice sites, A5SS: Alternative 5' splice sites, MXE: Mutually exclusive exons, RI: Retained introns, SE: Skipped exons, ALE: Alternative last exons. (B) Overlap of significant ALE events between SCAF4 KO, SCAF8 KO and dKO (C) Overlap of genes harboring significant ALE event in SCAF4 KO, SCAF8 KO and dKO. (D) Overlap of genes with an ALE event in dKO cells and genes with transcriptional readthrough in SCAF4 KOs. (E) UCSC genome browser view of identified cleavage sites (CS) and high-confidence cleavage sites (hcCS) in the UPF1-HOMER3 locus. (F-G) Position of identified of cleavage sites (F) and high-confidence cleavage sites (G) from 3'-Seq mapping to intergenic regions or within annotated Ensembl genes. The most significantly enriched motif in a 50 bp up- and downstream regions of both sets of cleavage-sites and the location relative to the cleavage site using MEME are shown to the left. (H) Outline of relative expression (RED) score calculation used to quantify changes in mRNA transcript cleavage based on 3'Seq data.

Fig 4: SCAF4 and SCAF8 Interact with Elongating RNAPII through Recognition of a Ser2-Ser5 Bi-phosphorylated CTD(A) IP of SCAFs from the chromatin fraction of cells expressing (or not) FLAG-tagged SCAF proteins.(B) SCAF binding assays using CTD peptides consisting of 4 heptad repeats, with phosphorylation at the indicated positions. NaCl washes (0.05, 0.2, 0.4, 0.8, and 1 M) were used to test the strength of binding.(C and D) Network analysis, using Cytoscape, of SCAF4 (C) and SCAF8 (D) interaction partners detected by label-free mass spectrometry of FLAG-IPs, using cells not expressing epitope-tagged protein as controls. The width of a connecting line represents average significance, while the width of the edge around the node represents average t test difference between the SCAF4 or SCAF8 IP and their control IP.(E) Western blot analysis of factors co-precipitating with FLAG-SCAF4 or FLAG-SCAF8.See also Figures S2 and S3 and Table S1.

Fig 5: TT-Seq and DRB/TT-Seq in SCAF4 and/or SCAF8 KOs, Related to Figures 3 and 4(A) Outline of TT-Seq to measure nascent RNA transcription. Cells were labeled with 1 mM 4SU for 15 min. Total RNA isolated, fragmented to 200-500 nt, 4SU residues biotinylated and purified using streptavidin beads to separate pre-existing non-labeled RNA from biotinylated 4SU-labeled newly synthesized RNA. 4SU-RNA is used for strand-specific library preparation and high-throughput sequencing. (B) Principal component analysis (PCA) of the 24 individual TT-Seq samples. SCAF4 KO cells with transcriptional readthrough are highlighted. Each cell line was sequenced from two biological replicate experiments, resulting in 4X WT samples (2 cell lines x 2 biological replicates), 6X SCAF4 KO samples (3 cell lines x 2 biological replicates, 6X SCAF8 KO samples (3 cell lines x 2 biological replicates) and 8X dKO samples (4 cell lines x2 biological replicates). (C) TT-Seq profiles around the TSS and TES of protein coding genes. (D) Metagene profiles for lncRNA, snoRNA and snRNA including the 5 kb upstream and downstream regions. (E) Metagene profile for the 1000 genes with the highest variation of readthrough ratios between any CRISPR group (SCAF4 KO (4KO), SCAF8 KO (8KO) or dKO and WT. (F) Overlap of readthrough genes between the three SCAF4 KO cells lines (SCAF4 KO, dKO3/GFP-SCAF8 and dKO4/GFP-SCAF8) with a relative readthrough ratios above 1.5 in two biological replicates of the indicated cell line (SCAF4 KO versus SCAF4 KO/GFP-SCAF4, dKO3 versus dKO3 versus dKO3/GFP-SCAF8 and dKO4 versus dKO4/GFP-SCAF8). (G) Correlation plot of readthrough ratios for the top 1000 genes with the largest coefficients of variation in their readthrough ratios across all individual 24 samples. WT (green), SCAF4 KO (red), SCAF8 KO (purple) and dKO (blue) cell lines are indicated. (H) RNAPII elongation wave peak positions for individual genes (> 60 kb) calculated from DRB/TT-Seq data following DRB release. Please note that the variation of the 40 min wave peak positions in some cases are caused by RNAPII termination downstream of the TES.