Fig 2: PCF11 Enhances Transcription Termination Genome-wide(A) Genomic profile of TSC22D1. Gray shading: termination window in control cells (siLUC, black). PCF11 depletion (siPCF11) is depicted in red. For chrRNA-seq and mNET-seq only sense strand is shown; ChIP is not strand specific. In all profiles, numbers in brackets indicate the viewing range (rpm).(B–D) Metagene analysis of total Pol II ChIP-seq (B), T4ph mNET-seq (C), and CPSF73 ChIP-seq (D) in cells ± siPCF11 on pc genes >5 kb long with PAS separated by >6 kb from the nearest gene on the same strand (n = 8,389).

Fig 3: Loss of MBLAC1 leads to 3' end processing defects in replication dependent histone pre-mRNA.(A) Western blots showing the knockdown efficiency of siRNA treatments for MBLAC1 and CPSF73 in synchronised HeLa cells (using ß-actin as a control). (B, C) ChrRNA-seq analyses from one of two biological replicates (Set2), on GAPDH or selected RD histone (HIST1H2BC) genes (UCSC Genome Browser) (Kent et al., 2002), following MBLAC1 or CPSF73 depletion. Arrows indicate gene bodies and transcription termination defect orientation. (D) Real-time qPCR quantification of 3' end transcription termination defect following MBLAC1 and CPSF73 depletion on genes as in (b, c). Relative abundance (RA) of unprocessed RNA was normalised to the abundance of GAPDH mRNA (black), or to that of the corresponding histone gene body (white) (Table 2). Error bars represent SEM from four biological replicates. Asterisks indicate the statistical significance observed using the two-tailed t-test (**p<0.01). Abbreviations: GB, gene body; RT, read through; TES, transcription end site. (E) Real Time-qPCR quantification of 3' end transcription termination defect in the MBLAC1 CRISPR/Cas9-mediated stable knockdown (KD) compared to wt HeLa cells. The relative abundance (RA) of unprocessed RNA was normalised to GAPDH mRNA (black), or to the corresponding histone gene body abundance (white) (Table 2). Error bars represent SEM from four biological replicates. Asterisks indicate the statistical significance observed using the two-tailed t-test (*,p<0.05, **, p<0.01, ***, p<0.001). Unlabeled variations were considered likely not statistically relevant. (F) RNA-Chromatin immunoprecipitation (RNA-ChIP) analyses investigating the recruitment of MBLAC1 on histone RNA. RNA-ChIP signals are shown for MBLAC1 and FLASH in the MBLAC1 C-terminal FLAG knock in HeLa cells; the FLAG background signal is shown in wild-type cells used as control for comparison. Note that both MBLAC1 and FLASH signals are comparable on normal protein coding RNA (GAPDH). Error bars represent SEM from three (MBLAC1) or two (FLASH) biological replicates. (G) RNA-ChIP input samples relative comparison. Histone mRNA relative abundance (in both wild-type and MBLAC1 C-terminal FLAG knock HeLa cells) was normalised to the abundance of GAPDH mRNA in the corresponding sample. The observed results imply that the RD histone mRNA levels are not affected (within detection limits) by the tagging of MBLAC1. Error bars represent SEM from three biological replicates.

Fig 4: INTS11 catalyzes the 3′ end cleavage of protein-coding transcripts at canonical termination sites.(A) 3′ RNA-seq Genome Browser example of the IRT gene DELE1 in WT INTS11 and E203Q INTS11 rescue cells. (B) CM of canonical versus downstream termination site usage in WT INTS11 rescue cells. (C) CM of canonical versus downstream termination site usage in E203Q INTS11 rescue cells. (D) Motif analysis at canonical 3′ end and downstream extended transcript 3′ end. A window of −/+150 nt from the peak was used for the analysis; top 5 motifs were shown. (E) CPSF73 ChIP-seq profiles at 3′ end in the downstream region of IRT genes. Profiles without (blue line) and with INTS11 shRNA induction (red line). (F) CSTF64 ChIP-seq profiles at 3′ end in the downstream region of IRT genes. Profiles without (blue line) and with INTS11 shRNA induction (red line).

Fig 5: Role of FLASH in recruiting polyadenylation factors of the HCC to semi-recombinant holo U7 snRNP and its catalytic activity. (A) Amino acid sequence of the N-terminal region of FLASH. The essential LDLY motif is boxed and the start points of the Δ51 and Δ61 deletions are indicated with the arrows. The region that binds Lsm11 is underlined. (B) Proteins of mouse nuclear extract that associate with the in vitro assembled core U7 snRNP either lacking (lane 1) or containing (lane 2) recombinant Δ51 FLASH. The complexes were immobilized on SA beads via mH2a/2m-3′B pre-mRNA, as described in the legend for Figure 2A, and bound proteins were visualized by silver staining. (C) In vitro assembled core U7 snRNP was bound to either 139N FLASH or its inactive deletion mutant lacking the LDLY motif, Δ61. The resultant complexes were next bound in the presence of FLAG-tagged SLBP to radioactively labeled mH2a/2m-3′B pre-mRNA, immobilized on SA beads and incubated with mouse nuclear extract to reconstitute semi-recombinant holo U7 snRNP. The presence of the HCC components (symplekin, CPSF100, CPSF73 and CstF64), SLBP and each of the two forms of FLASH, each N-terminally tagged with GST, was monitored by Western blotting (top panel) and silver staining (bottom panel). (D) Small aliquots of immobilized processing complexes containing either 139N or Δ61 FLASH were incubated at 0°C and 32°C to evaluate their ability to support endonucleolytic cleavage of 5′-labeled mH2a/2m-3′B pre-mRNA. (E–I) In vitro assembled core SupU7 snRNP was bound to Δ61 or 139N FLASH. The complexes were purified by size exclusion chromatography, analyzed by Western blotting (panel E) and tested for the ability to support processing of H2a/Ys (panel F), ΔSL/Ys (panel G) and DCP/Ys (panels H and I) in a mouse nuclear extract.