Journal: Molecular Cell

Article Title: A direct interaction between CPF and RNA Pol II links RNA 3′ end processing to transcription

doi: 10.1016/j.molcel.2023.11.004

Figure Lengend Snippet: CPF and APT interact directly with RNA Pol II (A) Pull-down assay of CPF or APT using immobilized StrepII (SII)-tagged RNA Pol II (Rpb3-His-SII). Input and bound proteins were analyzed on SDS-PAGE. Labels show APT subunits (purple), core CPF subunits (black), and RNA Pol II (blue). The experiment was repeated twice. (B) (Top) Analytical size exclusion chromatography profiles of RNA Pol II (blue) and APT (purple), loaded separately or after incubation (black). APT with RNA Pol II-ΔCTD is in gray. (Bottom) SDS-PAGE (cropped) of the fractions indicated by a red line. The black dashed box indicates the migration position of the RNA Pol II-APT complex. Gels are outlined according to colors of chromatograms. (C) SDS-PAGE showing 3C protease cleavage of the Rpb1 CTD to make RNA Pol II-ΔCTD. (D) Analytical size exclusion chromatography experiment, as in (B), of RNA Pol II incubated with the Ref2-Glc7-Swd2 subcomplex of APT. See also Figures S1 and .

Article Snippet: Anti-RNA Pol II pSer5 (clone 3E8) , Antibody Core Facility - Helmholtz-Zentrum Munich , N/A.

Techniques: Pull Down Assay, SDS Page, Size-exclusion Chromatography, Incubation, Migration

Journal: Molecular Cell

Article Title: A direct interaction between CPF and RNA Pol II links RNA 3′ end processing to transcription

doi: 10.1016/j.molcel.2023.11.004

Figure Lengend Snippet: Cryo-EM analysis reveals RNA Pol II homodimers in the presence of Ref2-Glc7-Swd2 (A) 3D reconstruction of RNA Pol II-APT (gray transparent surface) with a model of RNA Pol II (PDB: 5C4X ; blue ribbon) rigid-body fit into the map. DNA is in yellow, and RNA is in red. Density not accounted for is indicated. Arrows point at the RNA exit channel. (B and C) Selected 2D class averages of (B) DNA-RNA-loaded RNA Pol II with Ref2-Glc7-Swd2 from 850,000 particles and (C) DNA-RNA-loaded RNA Pol II from 400,000 particles. Classes are ordered by the number of particles in each class, from the most populated (1) to the least populated (9). (D) Composite map of the RNA Pol II dimer after signal subtraction and focused 3D refinement of the individual monomers (global resolution 3.6 Å). Monomers are colored in blue and gray. The DNA-RNA scaffold is in yellow. Anisotropy is due to preferred particle orientation on the grid. See also Figures S3 – .

Article Snippet: Anti-RNA Pol II pSer5 (clone 3E8) , Antibody Core Facility - Helmholtz-Zentrum Munich , N/A.

Techniques: Cryo-EM Sample Prep

Journal: Molecular Cell

Article Title: A direct interaction between CPF and RNA Pol II links RNA 3′ end processing to transcription

doi: 10.1016/j.molcel.2023.11.004

Figure Lengend Snippet: Cryo-EM data collection and processing

Article Snippet: Anti-RNA Pol II pSer5 (clone 3E8) , Antibody Core Facility - Helmholtz-Zentrum Munich , N/A.

Techniques:

Journal: Molecular Cell

Article Title: A direct interaction between CPF and RNA Pol II links RNA 3′ end processing to transcription

doi: 10.1016/j.molcel.2023.11.004

Figure Lengend Snippet: RNA Pol II dimerizes via the stalk protein Rpb7 (A) Two copies of monomeric RNA Pol II rigid-body fit into the dimeric RNA Pol II cryo-EM density. Monomer 1, blue; monomer 2, cyan; DNA, yellow. The magenta and green boxes on the model indicate the close-up views of the RNA Pol II dimer interfaces in (B) and (C), respectively. (B) Hydrophobic interaction between the Rpb7 subunits from each monomer. (C) Interaction between Rpb1 from monomer 1 with Rpb4 from monomer 2. (D–F) SDS-PAGE and cryo-EM analysis of RNA Pol II dimerization. 2D class averages were generated from 400,000 particles for RNA Pol II-Δstalk (D), RNA Pol II-Δstalk with recombinant wild-type (WT) stalk (E), or RNA Pol II-Δstalk with recombinant mutant stalk (F). Selected 2D classes are ordered by the number of particles per class, and dimeric classes are highlighted in red. Asterisks denote mutant stalk proteins. (G) Dynamic light scattering analysis on wild-type (dark gray) or Δstalk RNA Pol II (cyan). The hydrodynamic radius (R h ) is plotted as a function of RNA Pol II concentration. Black bars represent mean ± SD on 40 acquisitions for each concentration. The brackets on the right represent the increase in R h from the lowest to the highest concentration. See also Figure S7 and .

Article Snippet: Anti-RNA Pol II pSer5 (clone 3E8) , Antibody Core Facility - Helmholtz-Zentrum Munich , N/A.

Techniques: Cryo-EM Sample Prep, SDS Page, Generated, Recombinant, Mutagenesis, Concentration Assay

Journal: Molecular Cell

Article Title: A direct interaction between CPF and RNA Pol II links RNA 3′ end processing to transcription

doi: 10.1016/j.molcel.2023.11.004

Figure Lengend Snippet: Dephosphorylation promotes RNA Pol II dimerization (A) Selected 2D class averages of RNA Pol II-ΔCTD from 400,000 particles, ordered by the number of particles in each class. A dimeric class is highlighted in red. (B) Dynamic light scattering analysis on wild-type (dark gray; replotted from Figure 3 G) or RNA Pol II-ΔCTD (green). Black bars represent mean ± SD on 40 acquisitions for each concentration. The brackets on the right represent the increase in R h between the lowest and highest concentrations for each condition. (C) Analysis of RNA Pol II dimerization by negative stain EM. The percentage of dimeric particles per micrograph is plotted. For each RNA Pol II treatment, 60 (left) or 50 (middle and right) micrographs were acquired. The experiments in the left and middle panels were performed twice, once as a “double-blind” experiment. Black bars represent the mean ± SD. Pairwise comparisons are shown as indicated, where ∗∗∗∗ p < 0.0001 by one-way ANOVA Tukey’s test. (D) Schematic of regions used to calculate the RNA Pol II retention index. TSS, transcription start site; TES, transcription end site; pc, protein-coding. (E) Scatter plot of the log 2 fold change in RNA Pol II retention index versus the log 2 -fold change in RNA Pol II signal across the transcribed unit for genes with annotated transcription start and end sites (5,357 genes). Data points highlighted in red correspond to genes with RNA Pol II retention at the 3′ end (clusters 2–4 from the subset of data analyzed in F). The percentage of genes within each retention index bracket is shown. (F) k-means clustering and heatmap of RNA Pol II occupancy beyond the transcription end site (TES) at mRNA genes with RNA Pol II signal ≥0.5 in both wild-type (WT) and Rpb7 QHF cells. Clusters 2–4 show RNA Pol II retention in Rpb7 QHF cells compared with WT. See also Figures S8 and .

Article Snippet: Anti-RNA Pol II pSer5 (clone 3E8) , Antibody Core Facility - Helmholtz-Zentrum Munich , N/A.

Techniques: De-Phosphorylation Assay, Concentration Assay, Staining

Journal: Molecular Cell

Article Title: A direct interaction between CPF and RNA Pol II links RNA 3′ end processing to transcription

doi: 10.1016/j.molcel.2023.11.004

Figure Lengend Snippet: The Ref2 subunit of CPF and APT is a regulatory subunit of Glc7 (A) Schematic of the Glc7-Ref2 348–406 chimeric protein used for crystallization. Orange, Glc7; blue, Ref2 348–406 ; gray, glycine-serine linker; dark blue, two SPTYSPS RNA Pol II CTD repeats. Black lines indicate the regions visible in the crystal structure. (B) Cartoon representation of the Glc7-Ref2 348–406 crystal structure in two orientations and close-up views of the interactions of Ref2 residues 358–362 (left) and of the intermolecular β sheet formed between Glc7 and Ref2, including interaction details for the hydrophobic pair (ΦΦ) (right). Two manganese ions and a phosphate group are shown in ball-and-stick. Coordination waters are in red. The N and C termini are indicated. (C) View of the interaction interface between Ref2 348–406 in cartoon and Glc7 in surface representation. The inset shows how Ref2 binds Glc7 through the conserved “RVxF” motif. (D) Sequence alignment of yeast Ref2 and putative orthologs from Rattus norvegicus ( Rn ) and Homo sapiens ( Hs ). The Ref2 “I/L-x-R-x-G-K/R” motif is enclosed in a light blue box. The RVxF and downstream ΦΦ motifs shared among PP1-regulators are highlighted in light blue. (E) Immunoblots (top, anti-FLAG; bottom, anti-α-tubulin) of Ref2-mAID cells before or after addition of 1 mM auxin (IAA). Ref2-mAID is degraded within 15 min of adding auxin. The anti-FLAG antibody cross-reacts with a protein ( ∗ ) also present in the wild-type parent strain (final lane). n = 3. (F) Growth curves of Ref2-mAID (left) or Ref2-mAID cells co-expressing a triple-point mutant of Ref2 (Ref2 mut : I372D, F374K, Y384E). Cells were grown with 1 mM auxin (IAA) or an equivalent volume of DMSO. Dotted line, mean OD 600 of biological replicates (n = 3); shaded area, standard deviation of the mean. See also Figure S10 .

Article Snippet: Anti-RNA Pol II pSer5 (clone 3E8) , Antibody Core Facility - Helmholtz-Zentrum Munich , N/A.

Techniques: Crystallization Assay, Sequencing, Western Blot, Expressing, Mutagenesis, Standard Deviation

Journal: Molecular Cell

Article Title: A direct interaction between CPF and RNA Pol II links RNA 3′ end processing to transcription

doi: 10.1016/j.molcel.2023.11.004

Figure Lengend Snippet: Ref2 is required for the APT interaction with RNA Pol II (A) Pull-down assay of APT and APT-ΔRef2 with RNA Pol II immobilized on StrepTactin beads. SII, StrepII-tagged protein; APT subunits, purple; RNA Pol II subunits, blue; asterisk, degradation product. APT-ΔRef2 was obtained after Ref2 was degraded by contaminating proteases during purification of APT. (B) Analytical size exclusion chromatography of RNA Pol II and Ref2-ΔIDR-Glc7-Swd2. The fractions indicated by a red line were analyzed on the SDS-PAGE below. See also Figure S11 .

Article Snippet: Anti-RNA Pol II pSer5 (clone 3E8) , Antibody Core Facility - Helmholtz-Zentrum Munich , N/A.

Techniques: Pull Down Assay, Purification, Size-exclusion Chromatography, SDS Page

Journal: Molecular Cell

Article Title: A direct interaction between CPF and RNA Pol II links RNA 3′ end processing to transcription

doi: 10.1016/j.molcel.2023.11.004

Figure Lengend Snippet: Proposed model for the role of CPF in transcription termination (A) CPF and APT can bind elongating RNA Pol II to monitor RNA as it emerges from the RNA exit channel. PAS sequence, orange box; cleavage site, gray box; nascent RNA, dark red; yellow circles, phosphorylation. Transcription elongation factors Spt4/5 are also shown. (B) After the PAS is transcribed, CPF-mediated pre-mRNA cleavage and RNA Pol II CTD-dephosphorylation are activated. As a result, the newly exposed 5′ end still attached to RNA Pol II is degraded by the torpedo exonuclease Rat1 (left), and RNA Pol II dephosphorylation triggers an allosteric event (RNA Pol II dimerization), which displaces transcription factors, or domains of transcription factors (right). Thus, CPF may convert RNA Pol II into a termination-competent complex. It remains unclear whether the second RNA Pol II is also transcribing. See also Figure S12 .

Article Snippet: Anti-RNA Pol II pSer5 (clone 3E8) , Antibody Core Facility - Helmholtz-Zentrum Munich , N/A.

Techniques: Sequencing, Phospho-proteomics, De-Phosphorylation Assay

Journal: Molecular Cell

Article Title: A direct interaction between CPF and RNA Pol II links RNA 3′ end processing to transcription

doi: 10.1016/j.molcel.2023.11.004

Figure Lengend Snippet:

Article Snippet: Anti-RNA Pol II pSer5 (clone 3E8) , Antibody Core Facility - Helmholtz-Zentrum Munich , N/A.

Techniques: Virus, Recombinant, Protease Inhibitor, Western Blot, Isolation, Reverse Transcription, SYBR Green Assay, Expressing, CRISPR, Mutagenesis, Software

Journal: Nature Communications

Article Title: The host RNA polymerase II C-terminal domain is the anchor for replication of the influenza virus genome

doi: 10.1038/s41467-024-45205-2

Figure Lengend Snippet: WSN FluPol binding to huANP32A ( A ) and WSN FluPol oligomerisation ( B ) in the presence of increasing amounts of pS5 CTD, as assessed using split-luciferase-based complementation assays. Plasmids encoding mCherry or mCherry fused to CTD-WT (dark grey bars) or CTD-S5A (serine five residues replaced with alanines, light grey bars) were co-transfected in increasing amounts (mean ± SD, n = 3, ** p < 0.002, *** p < 0.001, two-way ANOVA; Sidak’s multiple comparisons test). Luminescence signals are represented as fold-changes compared to untagged mCherry co-expression. In ( B ), cell lysates were analysed by western blot using the indicated antibodies ( n = 1). C 51-mer vRNA template (v51_mut_S) derived from segment 4 of A/Zhejiang/DUID-ZJU01/2013(H7N9)/KJ633805 and used in in vitro replication activity assays. The vRNA 5’end (1–30) and 3′ end (31–51) are coloured in pink and gold, respectively, with introduced mutations in red. The theoretical de novo full-length replication product is coloured in light blue (1–33) and dark blue (35–51), with U34 in red. The expected stalled elongation state is schematically represented. de novo replication activity assays of Zhejiang-H7N9 FluPol using v51_mut_S, in the presence of either 3 NTPs (AUG) or 4 NTPs (AUGC), with or without pS5 CTD(6mer) and huANP32A full-length ( D , n = 3) or deletion mutants ( E , n = 3). Tentative full-length and stalled replication products are indicated by an arrow. LRR leucine-rich repeat, LCAR low complexity acidic region, NLS nuclear localisation signal. Nts: molecular weight marker. F WSN and Anhui-H7N9 vRNPs were reconstituted in ANP32AB KO cells with a model Fluc-vRNA, and co-expressed with the indicated huANP32A proteins (V5-tagged and fused to SV40-NLS). Luminescence signals are represented as a percentage of huANP32A FL. (mean±SD, n = 3, *** p < 0.001, two-way ANOVA; Sidak’s multiple comparisons test). G Lysates of ANP32AB KO cells transiently expressing the indicated huANP32A proteins were analysed by western blot using an anti-V5 antibody ( n = 1). H , I RNA-sequencing of Zhejiang-H7N9-4M FluPol de novo replication products in the presence of all NTPs, the pS5 CTD(6mer) and huANP32A. H The number of reads is plotted according to the recurrence of the exact 5’ cRNA motifs indicated on the left. Reads that do not encompass these motifs are plotted as ‘Other’. I The percentages of full-length replication products are plotted according to the 5’ terminal nucleotide indicated on the left. Source data are provided as a file.

Article Snippet: Proteins were separated by SDS-PAGE using NuPAGETM 4-12% Bis-Tris gels (Invitrogen) and transferred to nitrocellulose membranes which were incubated with primary antibodies directed against pS5 CTD (clone 3E8, Active Motif, 1:1000), mCherry (26765-1-AP, Proteintech, 1:000), V5 (SV5-Pk1, Thermo Fisher, 1:5000), Tubulin (B-5-1-2, Sigma-Aldrich, 1:10000), PA (1:2500), PB2 (GTX125925, GeneTex, 1:5000), ANP32A (AV40203, Sigma-Aldrich, 1:2500), ANP32B (EPR14588, AbCam, 1:2500) and subsequently with HRP-tagged secondary antibodies (Jackson Immunoresearch).

Techniques: Binding Assay, Luciferase, Transfection, Expressing, Western Blot, Derivative Assay, In Vitro, Activity Assay, Molecular Weight, Marker, RNA Sequencing

Journal: Nature Communications

Article Title: The host RNA polymerase II C-terminal domain is the anchor for replication of the influenza virus genome

doi: 10.1038/s41467-024-45205-2

Figure Lengend Snippet: Cryo-EM structures data collection, refinement and validation statistics

Article Snippet: Proteins were separated by SDS-PAGE using NuPAGETM 4-12% Bis-Tris gels (Invitrogen) and transferred to nitrocellulose membranes which were incubated with primary antibodies directed against pS5 CTD (clone 3E8, Active Motif, 1:1000), mCherry (26765-1-AP, Proteintech, 1:000), V5 (SV5-Pk1, Thermo Fisher, 1:5000), Tubulin (B-5-1-2, Sigma-Aldrich, 1:10000), PA (1:2500), PB2 (GTX125925, GeneTex, 1:5000), ANP32A (AV40203, Sigma-Aldrich, 1:2500), ANP32B (EPR14588, AbCam, 1:2500) and subsequently with HRP-tagged secondary antibodies (Jackson Immunoresearch).

Techniques: Biomarker Discovery, Microscopy

Journal: Nature Communications

Article Title: The host RNA polymerase II C-terminal domain is the anchor for replication of the influenza virus genome

doi: 10.1038/s41467-024-45205-2

Figure Lengend Snippet: A Left: Cartoon representation of Flu A Pol Zhejiang-H7N9-4M in the intermediate conformation (obtained in this study) as previously described , , bound to pS5 CTD. Middle: Flu A Pol Zhejiang-H7N9-4M in a replicase-like conformation bound to pS5 CTD. Right: Flu A Pol replicase conformation from A/duck/Fujian/01/2002(H5N1) ( PDB: 6QPF ) . FluPols are aligned on the PB1 subunit. Flu A Pol Zhejiang-H7N9-4M PA-ENDO remains in a transcriptase conformation (PA-ENDO(T)). In Flu A Pol Fujian-H5N1 structure, it rotates and interacts with the PB2-NLS domain (PA-ENDO(R)). PA is coloured in green, PB1 in light grey, PB2-N in dark red, PB2-CBD in orange, PB2 mid-link in purple, PB2 627 in plum, PB2 NLS in salmon. The pS5 CTD is coloured in red, displayed as surface, with discontinuity between sites 1A/2A shown as a dotted line. PB2-627/NLS domains flexibility is highlighted as a dotted circle. RNAs are displayed as surfaces. The 5′ vRNA end is coloured in pink, the 3′ vRNA end in yellow. Flexible nts are represented as solid line. B Cartoon representation of Flu A Pol Zhejiang-H7N9-4M structure in the pre-initiation state mode A. Colour code is identical to ( A ). PB2 C-terminal domains (PB2-C) are flexible, highlighted as a dotted circle. C Close-up view on the 3′ vRNA end in Flu A Pol Zhejiang-H7N9-4M active site. The Coulomb potential map of the template is shown. 3′-U1 remains unseen in the map. The 3′-G3 is in the +1 active site, highlighted by a dotted line. The priming loop is coloured in red, the palm domain in orange with the catalytic aspartic acids displayed, coordinating a Mg 2+ ion, in green. The motif F is coloured in blue. D Schematic representation of the 3′ vRNA end terminal nucleotides active site position, as seen in ( C ). E Cartoon representation of Flu A Pol Zhejiang-H7N9-4M in stalled elongation state. The colour code is identical to A. PB2-C domains are flexible. The cRNA de novo replication product is displayed as surface when visible or as a dotted line when flexible. The 3′ vRNA end is bound to the secondary site. F Close-up view on the pS5 CTD interaction with Flu A Pol Zhejiang-H7N9-4M PA-C domain. FluPol residues interacting with the pS5 CTD are shown. Each CTD pS5 repeat is indicated.

Article Snippet: Proteins were separated by SDS-PAGE using NuPAGETM 4-12% Bis-Tris gels (Invitrogen) and transferred to nitrocellulose membranes which were incubated with primary antibodies directed against pS5 CTD (clone 3E8, Active Motif, 1:1000), mCherry (26765-1-AP, Proteintech, 1:000), V5 (SV5-Pk1, Thermo Fisher, 1:5000), Tubulin (B-5-1-2, Sigma-Aldrich, 1:10000), PA (1:2500), PB2 (GTX125925, GeneTex, 1:5000), ANP32A (AV40203, Sigma-Aldrich, 1:2500), ANP32B (EPR14588, AbCam, 1:2500) and subsequently with HRP-tagged secondary antibodies (Jackson Immunoresearch).

Techniques:

Journal: Nature Communications

Article Title: The host RNA polymerase II C-terminal domain is the anchor for replication of the influenza virus genome

doi: 10.1038/s41467-024-45205-2

Figure Lengend Snippet: A Cartoon representation of the pS5 CTD-bound to FluPol Zhejiang-H7N9 in site 2A ( PDB: 7Z4O ) . PA subunit is coloured in green, pS5 CTD in red. PA K289-C489 residues are displayed. Putative hydrogen bonds are drawn as yellow dashed lines. Distances are indicated. B Cartoon representation of Flu A Pol Zhejiang-H7N9 PA K289A + C489R (obtained in this study). The Coulomb potential map of PA/K289A-C489R is shown. C Superposition of the pS5 CTD, extracted from the structure shown in ( A ), with the Flu A Pol Zhejiang-H7N9 PA K289A + C489R structure shown in ( B ). The putative hydrogen bond between PA C489R and pS5 is shown. Distance is indicated. D – I Phenotypes associated with the WSN FluPol PA K289A primary mutation and PA C489R second-site mutation. D Plaque phenotype of recombinant WSN mutant viruses produced by reverse genetics ( n = 2), analyzed as in Fig. (see Fig. ) (#) pinhead-sized plaques. E WSN FluPol activity was measured by vRNP reconstitution in HEK-293T cells, using a model Fluc-vRNA. Luminescence signals are represented as a percentage of PA WT. F WSN vRNPs were reconstituted in HEK-293T cells using the NA vRNA segment. The steady-state levels of NA mRNA and vRNA were quantified by strand-specific RT-qPCR , normalised to GAPDH by the 2 −ΔΔCT method and are presented as ratios of mRNA to vRNA levels relative to PA WT (RNA levels are shown in Fig. ). G WSN FluPol binding to the CTD was assessed using a split-luciferase-based complementation assay. Luminescence signals are represented as a percentage of PA WT. H Accumulation levels of cRNA and vRNA in a vRNP reconstitution assay were determined by strand-specific RT-qPCR as in ( F ). Ratios of cRNA to vRNA levels relative to PA WT are shown (RNA levels are shown in Fig. ). I huANP32A-binding to WSN FluPol was determined as in ( G ). (mean ± SD, n = 3, 3, 3, 3, 5, * p < 0.033, ** p < 0.002, *** p < 0.001, one-way ANOVA; Dunnett’s multiple comparisons test). Source data are provided as a file.

Article Snippet: Proteins were separated by SDS-PAGE using NuPAGETM 4-12% Bis-Tris gels (Invitrogen) and transferred to nitrocellulose membranes which were incubated with primary antibodies directed against pS5 CTD (clone 3E8, Active Motif, 1:1000), mCherry (26765-1-AP, Proteintech, 1:000), V5 (SV5-Pk1, Thermo Fisher, 1:5000), Tubulin (B-5-1-2, Sigma-Aldrich, 1:10000), PA (1:2500), PB2 (GTX125925, GeneTex, 1:5000), ANP32A (AV40203, Sigma-Aldrich, 1:2500), ANP32B (EPR14588, AbCam, 1:2500) and subsequently with HRP-tagged secondary antibodies (Jackson Immunoresearch).

Techniques: Mutagenesis, Recombinant, Produced, Activity Assay, Quantitative RT-PCR, Binding Assay, Luciferase, Reconstitution Assay