Fig 2: 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 3: The 3′-UTR of histone H2AC18 pre-mRNA can assemble both histone and canonical 3′-mRNA-processing complexes. (A) Western blot analysis of complexes purified from HEK293-F nuclear extract (N.E.) with the H2A_4m RNA, prepared with different oligonucleotide concentrations, in the absence or presence of different control molecules (U7, aU7 + SL) or in the presence of the recombinant N-terminal FLASH (N-FLASH) (left subpanel); comparison of results obtained with HEK293-F and HeLa-S3, both using 40 nM H2A_4m (right subpanel). (B) Activity assay with a double-biotin wild-type (wt) H2AC18 3′-UTR sequence (structure on top), performed either in crude HEK293-F nuclear extract or with purified histone RNA-processing complex; cleavage products were purified and analyzed by northern blot and luminescent biotinylation detection; complex assembly and cleavage were blocked in the presence of aU7/SL oligonucleotides; the uncleavable GA-H2A was used as weight standard. (C, D) Differential analyses of protein abundances measured by MS-based quantitative proteomics: [H2A_4m] versus [H2A_4m + U7/SL] samples (C); [H2A_4m] versus [H2A_4m + aU7/SL] (D). The volcano plots represent the –log10(limma P-value) on the y-axis plotted against the log2(fold change) on the x-axis. Relevant identified proteins that are at least 2-fold more abundant with [H2A_4m] compared with [H2A_4m + U7/SL] or [H2A_4m + aU7/SL] are annotated in the zoom-in graphs (subpanels on the left); schematic representations of complex relative abundances are shown (lower subpanels). Abbreviations: CFIm, ceavage factor 1m module (including NUDT21, CPSF6 ands CPSF7 subunits); CPSF, cleavage and polyadenylation specificity factor; CstF, cleavage-stimulating factor module (including CSTF1, CSTF2, CSTF2T and CSTF3 subunits); FLASH, FLICE-associated huge protein; HCC/hCF, histone cleavage complex (SYMPK, CPSF100 and CPSF73 subunits)/human cleavage factor; hPSF, human polyadenylation specificity factor (including CPSF30, CPSF160, WDR33 and FIP1 subunits); PPase, phosphatase module (including SSU72, WDR82 and PP1CA/B/C/R10 subunits); SLBP, stem–loop-binding protein; SYMPK, Symplekin; U7 snRNP, U7 small nuclear ribonucleoprotein (including, besides U7 snRNA, LSM11, LSM10, SNRPD3/B/E/F subunits and the SNRRPG subunit not detected in our analysis).

Fig 4: Transcription termination pathways used by pol II transcribing human mRNA and snRNA genes. Termination factors that are common between both genes types are labelled in the same colour. Pol II CTD phosphorylation state is indicated as Phospho-Ser2 (S2P) and Phospho-Ser7 (S7P) (A). For protein-coding genes, the CPSF complex and the CstF complex recognize the poly A signal (AAUAAA) and U/GU-rich elements, respectively, as they emerge from the elongation complex. The CPSF-73 subunit, aided by CPSF-100, catalyzes cleavage at the poly A site (indicated by a lightning bolt) creating an entry site for the 5′–3′ exoribonuclease, Xrn2. Xrn2 degrades the downstream RNA product and displaces pol II. The CTD interacting domain of Pcf11 associates with the S2P form of the pol II CTD and bridges the polymerase with the nascent transcript, leading to pol II pausing and dismantling of the elongation complex. (B) The gene-specific PTF complex is recruited to the snRNA promoter and clears the transcription unit of nucleosomes to facilitate passage of pol II. The NELF complex is recruited at the end of the nucleosome-depleted region to terminate pol II where nucleosomes are encountered. CPSF-73 is not recruited to human snRNA genes but instead the Integrator complex directs cleavage upstream of the 3′ box processing element (indicated by a lightning bolt). The Int11 subunit is likely to catalyze the cleavage reaction with the help of Int9, as these subunits are homologous to CPSF-73 and CPSF-100, respectively. Thus, Integrator substitutes for the CPSF complex on snRNA genes. Ssu72 and Pcf11 are recruited to snRNA genes but do not have a major function in snRNA 3′-end processing. Instead, Ssu72 and Pcf11 act as transcription terminators for this gene class. CstF64 is also recruited, but its role in snRNA processing and transcription termination is not clear. Xrn2 is not implicated in snRNA transcription termination, but as cleavage upstream of the 3′ box would create an entry site for a 5′–3′ exoribonuclease, we cannot rule out that an alternative enzyme is recruited to promote termination.

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.