Fig 2: Early polyA Site Selection in SCAF4 and SCAF8 dKOs Leads to Production of Truncated Proteins(A) Distribution of pooled SCAF4 and SCAF8 consensus clusters (overlapping clusters found in =2 out of 6 samples from SCAF4 and/or SCAF8 experiments, but not in controls). Localization calculated from the cluster midpoint. Upstream, 2 kb upstream of TSS. Downstream, 2 kb downstream of TES.(B) Number of pooled SCAF4 and SCAF8 clusters found exclusively in exons or introns, as well as in clusters spanning an intron-exon (n = 2,271) or exon-intron (n = 3,772) boundary.(C) Meta-transcriptome profiles for SCAF binding clusters across mRNA transcripts.(D) Western blot of protein products produced from the shorter RNA isoforms of ZC3HAV1 and USP15 by antibodies raised against N-terminal peptides common to both isoforms.(E) Simple working model and summary of the main effects of SCAF4 and SCAF8 on polyA site selection and transcriptional termination.See also Figure S7.

Fig 3: 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 4: Early polyA Site Selection in SCAF4 SCAF8 dKOs Is Accompanied by Early Transcriptional Termination, Related to Figure 5Genome browser tracks showing nascent RNA (TT-Seq), mRNA-Seq and 3'-Seq for (A) RAD51C, (B) DNAJB6 and (C) CNTLN in dKO and WT cells. (D) Boxplot of nascent transcription (TT-Seq) for distal and proximal terminal exons in SCAF regulated genes (significant ALE event in WT versus dKOs, n = 340) and non-regulated genes with > 2 terminal exons (n = 6094). (E) Number of genes with less nascent (TT-Seq) transcription in their distal terminal exon compared to the proximal terminal exon for 10,000 randomly selected genes set (all n = 340) compared to the SCAF4 and SCAF8 regulated gene set (n = 340). (F-I) Genome browser tracks showing examples of genes with skipped exon (SE) events identified by MISO (affected exons as detected by MISO is indicated by green arrows) in COX10 (F), BCL11A (G), AKAP13 (H) and HP1BP3 (I). (J) Relative usage of intronic polyadenylation (IpA) sites identified by Singh et al., 2018 in SCAF4 KO (4KO), SCAF8 KO (8KO) and dKO (dKO). Only IpA sites with a total RPKM = 50 across all samples were considered.

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.