Fig 2: Transcriptome-wide analyses of CPSF–RNA interaction by iCLIP. (A) A tet-inducible GFP-CPSF160 expression HeLa cell line was untreated (−) or treated (+) with Dox, incubated with 4-SU, and UV-irradiated. (Lanes 1–3) Cell lysates were treated with RNase I before being subjected to immunoprecipitation (IP) with control (Ctrl) or anti-GFP antibodies. For samples in lanes 4–6, the immunoprecipitation samples were subjected to a second immunoprecipitation with anti-CPSF30 antibodies. All immunoprecipitation samples were 5′ labeled with 32P, resolved on a polyacrylamide gel, and visualized by phosphorimaging. (B) CPSF iCLIP tracks on the Rps12 and Atf4 genes. CstF64 iCLIP tracks based on data from Yao et al. (2012) were included for comparison. Each peak represents a unique cross-linking site. Nucleotide sequences around the iCLIP peaks are shown above the tracks. (C) Transcriptome-wide distribution of CPSF cross-linking sites relative to the cleavage sites (ends of transcripts). The gray line shows the distribution of the A(A/U)UAAA hexamer. The Y-axis is the percentage of CPSF cross-linking events or AAUAAA occurrence at that site over those in the entire region. (D) Transcriptome-wide distribution of CPSF cross-linking sites relative to AAUAAA (the underlined A corresponds to position 0 on the X-axis).

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: eIF4E Physically Associates with Some of the Cleavage Machinery and RNA Substrates for the Pathway(A) Endogenous eIF4E immunoprecipitated (IP) with CPSF1 and CPSF3 in nuclear lysates from U2OS cells. Fractionation controls for nuclear IP were Lamin and RNA polymerase II for the nucleus (N) and GAPDH and tubulin for the cytoplasm (C). Nc, nuclear lysate; S, supernatant.(B and D) Diagram of constructs, which is not to scale, with the position of primers to detect cleaved (reverse primer immediately downstream the cleavage site (C/A) + 13 thymine nucleotides, shown in pink (qRT-PCR; Figure S3C) and uncleaved LacZ transcripts (primers in red, forward used for both cleaved and uncleaved) (B). (D) depicts constructs used for studies into the deletion of the PAS region.(C) eIF4E-FLAG RIP using primers that bracket the cleavage site as used for the cleavage assay (uncleaved primers [red], exon “total” primers [green]). Results are relative to IgG controls. These findings indicate that, in the nucleus, eIF4E associated with uncleaved LacZ-4ESE RNA. (E) eIF4E-FLAG RIP from nuclear lysates shows PAS enriched association with LacZ-4ESE by 2-fold relative to LacZ-4ESE?PAS. As expected, eIF4E did not interact with VEGF RNA in nuclear lysates.Experiments were three biological replicates each performed in technical duplicates for (C) and (E) and three biological replicates for (A), each performed in technical triplicates. For qRT-PCR experiments, values are means ± SD with p values as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig 5: The integrity of the PAS sequence in H2AC18 is essential for the assembly of the canonical 3′-pre-mRNA-processing complex. (A) Assembly scheme of the canonical complex on the H2A_4m RNA: in purple is depicted the hPSF module (CPSF73, CPSF100 and SYMPK), mediating PAS recognition in the RNA sequence; elements predicted as not directly involved in the interaction (HDE* and other complex modules) are depicted in gray; a sequence alignment among H2A_4m, its double mutant GA-H2A and the mouse RNA mH2A* is reported below; in the H2A_4m sequence, the PAS is indicated in purple, mutated residues in 38G/40-H2A_4m are underlined and HDEs are indicated in green. (B) Western blot analysis of samples purified with H2A_4m bait RNA, in the absence or presence of PAS-containing control RNAs; the use of these molecules allowed blocking, in a selective manner, of the assembly on H2A_4m of the entire canonical complex via the hPSF module (scheme on the right). (C–E) Comparison by western blot of processing factor abundances in samples purified with different RNAs/conditions: H2A_4m versus GA-H2A (C), GA-H2A versus mouse RNA mH2A* (D) and GA-H2A in the absence or presence of aU7/SL RNAs (E); mutations in GA-H2A RNA disrupting the PAS sequence prevent the assembly of the entire canonical complex via the hPSF module (scheme below). In (B) and (E), the lanes separated by a black line come from different zones of the same membrane. In (C), nucleolin is used as a control protein to demonstrate the specificity of the PAS disruption effect on proteins belonging to the canonical mRNA-processing complex. Exposure time in (E) was increased, compared with (D), to better show the difference between samples without or with aU7 + SL (F) Western blot analyses with four antibodies of a sample purified with GA-H2A, DTSSP-cross-linked after elution from streptavidin–agarose beads and run in native PAGE. (G) Differential analysis of protein abundances in [H2A_4m] versus [GA-H2A] samples; relevant identified proteins that are at least 2-fold more abundant in [H2A_4m] samples compared with [GA-H2A] samples are annotated in the zoom-in graphs (subpanel on the left); a schematic representation of complex relative abundance is shown (lower subpanel). Abbreviations: CFIm, cleavage factor 1m module (including NUDT21, CPSF6 and 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).