Journal: bioRxiv

Article Title: The R2TP chaperone assembles cellular machineries in intestinal CBC stem cells and progenitors

doi: 10.1101/2019.12.19.882712

Figure Lengend Snippet: (A) Western blot analysis of preparations enriched for epithelial crypt cells from the jejunum of animals, sacrificed 6 days after the first tamoxifen injection. NOP58, PRP8, ATR and mTOR and TRRAP were detected with specific antibodies. Tubulin and GAPDH were used as loading controls. Each lane was loaded with the lysate obtained from one animal of the indicated genotype (6 animals per gel). Molecular weights are indicated on the right. See Supplemental Figure 4 for an expanded view of the membranes and signal quantification. (B) Images are tissue sections of small intestines stained by immunohistochemistry (IHC) for Rpb1, the large subunit of RNA polymerase II, from control Rpap3 flox/flox mice (blue frame, left panel), or VilCreER T2; Rpap3 flox/flox animals at day 6 (red frame, right panel). Note that the staining in stromal cells is nuclear in both wild-type and VilCreER T2 ; Rpap3 flox/flox animals (stromal cells do not express the Cre), while it becomes cytoplasmic in the mutant epithelium. The scale is identical for both pictures. Panels are representative for n=6 animals from three independent experiments. (C) Magnification of crypts from (B). The scale bar is identical for both images. (D) Schematic interpretation of the IHC in (B, C). In control epithelial cells, R2TP incorporates Rpb1 into RNA PolII, which is then imported into the nucleus. In the absence of Rpap3, neo-synthesized Rpb1 accumulates in the cytoplasm.

Article Snippet: Primary antibodies from Cell Signaling: ATM D2E2 (CST 2873S) rabbit at 1/1000, ATR E1S3S (CST 13934S) rabbit at 1/1000, mTOR (CST 2972S) rabbit 1/1000, p53 1C12 (CST2524S) mouse 1/1000; PRP8 (sc-30207, Santa Cruz) rabbit 1/200; from ABCAM: EFTUD2 (ab72456) rabbit 1/2000, GAPDH (ab8245 6C5) mouse 1/10000; and from Sigma: NOP58 (HPA018472) rabbit 1/100, RPAP3 (SAB1411438) rabbit 1/1000, Tubulin (I2G10) mouse 1/500, TRRAP 2TRR-1B3 (MABE1008) mouse 1/1000.

Techniques: Western Blot, Injection, Staining, Immunohistochemistry, Control, Mutagenesis, Synthesized

Journal: Nature Communications

Article Title: The HSP90/R2TP assembly chaperone promotes cell proliferation in the intestinal epithelium

doi: 10.1038/s41467-021-24792-4

Figure Lengend Snippet: a Images are tissue sections of small intestines stained by immunohistochemistry (IHC) for Rpb1, the catalytic subunit of RNA polymerase II, from control Rpap3 flox/flox mice (blue frame, left panel), or VilCreER T2 ; Rpap3 flox/flox animals at day 6 (red frame, right panel), with magnifications of crypts (scale bars used for the two magnification insets are identical between blue and red frames). Note that the staining in stromal cells is nuclear in both wild-type and VilCreER T2 ; Rpap3 flox/flox animals (stromal cells do not express the Cre) and control epithelium, while it becomes cytoplasmic in the mutant epithelium. Panels are representative for n = 6 animals from three independent experiments. Scale bar is 50 and 10 μm for insets, as shown in control panels. b Schematical interpretation of the micrographs in ( a ). In control epithelial cells (blue), R2TP incorporates Rpb1 into RNA PolII, which is then imported into the nucleus. In the absence of Rpap3 (red), neo-synthesized Rpb1 accumulates in the cytoplasm. c , d Western blot analysis of preparations enriched for epithelial crypt cells from the jejunum of animals, sacrificed 6 days after the first tamoxifen injection. NOP58, EFTUD2, and PRPF8 ( c ), mTOR, ATM, ATR, and TRRAP ( d ) were detected with specific antibodies. Tubulin and GAPDH were used as loading controls. Quantification of the signal ratios are indicated on top of each lane (average for the control ratios was arbitrarily set to 100). Each lane was loaded with the lysate obtained from one animal of the indicated genotype ( n = 3 per genotype). Similar results were obtained with animals from at least two independent experiments. Apparent molecular weights are indicated on the right. Source data are provided as a “ file”.

Article Snippet: TMA tissue sections were stained with a mouse monoclonal antibody raised against RPAP3 (proprietary 19B11 antibody at 1:10 dilution) or an antibody against human mTOR (clone 7C10, 1:100 dilution; overnight incubation; cat. number #2983; Cell Signaling).

Techniques: Staining, Immunohistochemistry, Control, Mutagenesis, Synthesized, Western Blot, Injection

Journal: Nature Communications

Article Title: The HSP90/R2TP assembly chaperone promotes cell proliferation in the intestinal epithelium

doi: 10.1038/s41467-021-24792-4

Figure Lengend Snippet: a Representative micrographs of colon sections stained by Periodic Acid Schiff stain (PAS, for labeling of goblet cells), IHC for Ki67 and CD44v6 (a marker of the colonic stem cells) and IF for p53, in controls Rpap3 flox/flox (left) and VilCreER T2 ; Rpap3 flox/flox mice (right) at day 8 ( n = 4 from at least two independent experiments). Scale bars are 50 μm. b Western blot analysis of colonic epithelial cells from animals, sacrificed 7 days after the first tamoxifen injection. mTOR, ATM, and tubulin were detected with specific antibodies. Quantification of the signal ratios are indicated on top of each lane (average for the control ratios was arbitrarily set to 100). Each lane was loaded with the lysate obtained from one animal of the indicated genotype (representative for n = 5 KO animals in one experiment). Molecular weights are indicated on the right. c Micrographs are tissue sections stained by IHC for cleaved caspase 3 in Rpap3 flox/flox (blue) and VilCreER T2 ; Rpap3 flox/flox (red) mice at day 6, representative for n = 4 from two different experiments. Total number of apoptotic cells at day 8 per surface (mm 2 ) of the jejunum for each mouse analyzed. Mean values with S.E.M are indicated for each experimental group. Unpaired two-tailed t test with Welch’s correction indicates significant difference between controls and VilCreER T2 ; Rpap3 flox/flox animals ( p = 0.0234, t = 2.625, df = 5). Scale bar is 50 μm. d Picture of organoids cultures. Crypts from small intestine (top) and colon (bottom) were prepared from Rpap3 flox/flox (blue) and VilCreER T2 ; Rpap3 flox/flox (red) mice 3 days after tamoxifen injection ( n = 3). Identical number of crypts were seeded and organoids culture were monitored every day. Organoids from control animals started budding after 72 h in culture for small intestine crypts and 96 h for colonic crypts. Organoids generated from VilCreER T2 ; Rpap3 flox/flox animals degenerated in culture before budding. Scale bar are 100 μm for all pictures and 20 μm for organoid insets. Source data are provided as a “ file”.

Article Snippet: TMA tissue sections were stained with a mouse monoclonal antibody raised against RPAP3 (proprietary 19B11 antibody at 1:10 dilution) or an antibody against human mTOR (clone 7C10, 1:100 dilution; overnight incubation; cat. number #2983; Cell Signaling).

Techniques: Staining, Labeling, Marker, Western Blot, Injection, Control, Two Tailed Test, Generated

Journal: bioRxiv

Article Title: The R2TP chaperone assembles cellular machineries in intestinal CBC stem cells and progenitors

doi: 10.1101/2019.12.19.882712

Figure Lengend Snippet: (A) Micrographs are tissue sections stained for Olfm, in the crypts from controls animals (left), or VilCreER T2 ; Rpap3 flox/flox animals 7 days after the first tamoxifen injection (right). (B) Staining for Olfm4 in the jejunum from controls (top panel) and VilCreER T2 ; Rpap3 flox/flox animals 6 to 8 days after the first tamoxifen injection. Scale is identical in all images (n=2 to 4 animals/time point from at least two independent experiments). (C) Schematic representation of a crypt from the small intestine, with CBC stem cells (in green) sandwiched between Paneth cells (in brown) and progenitors forming the TA on top (purple). (D) Micrographs are tissue sections stained for lysozyme, a specific marker of Paneth cells, in the jejunum of controls (top) and VilCreER T2 ; Rpap3 flox/flox mice from day 6 to day 8 (black arrows). Panels are representative for 2-3 animals/time-point from two independent experiments. The scale bar is identical in all pictures. (E) Micrographs are tissue sections stained for cleaved caspase 3 (cleaved cas3), a marker of apoptosis, in the jejunum. In control animals, cleaved cas3 + cells (brown arrows) are mainly detected at the tip of the villi, as a result of epithelium turnover (top panel). In the jejunum from VilCreER T2 ; Rpap3 flox/flox animals, at day 6 and 7, cleaved caspase 3 + cells were detected within the crypts(brown arrows). Panels are representative from 2 to 4 animals/time point, from two independent experiments. The graph on top of the column shows the total number of apoptotic cells at day 6, divided by the surface of the jejunum for each mouse analyzed. Mean values with S.E.M are indicated for each experimental group. Unpaired two-tailed t-test with Welch’s correction indicates significant difference between controls and VilCreER T2 ; Rpap3 flox/flox animals (p=0, 0384; t=3,538, df=3, n=4). (F) Micrographs are tissue sections stained for p53 by immunofluorescence in controls (left) and VilCreER T2 ; Rpap3 flox/flox mice (right) at day 6 (n=4). Dapi: nuclei.

Article Snippet: Primary antibodies from Cell Signaling: ATM D2E2 (CST 2873S) rabbit at 1/1000, ATR E1S3S (CST 13934S) rabbit at 1/1000, mTOR (CST 2972S) rabbit 1/1000, p53 1C12 (CST2524S) mouse 1/1000; PRP8 (sc-30207, Santa Cruz) rabbit 1/200; from ABCAM: EFTUD2 (ab72456) rabbit 1/2000, GAPDH (ab8245 6C5) mouse 1/10000; and from Sigma: NOP58 (HPA018472) rabbit 1/100, RPAP3 (SAB1411438) rabbit 1/1000, Tubulin (I2G10) mouse 1/500, TRRAP 2TRR-1B3 (MABE1008) mouse 1/1000.

Techniques: Staining, Injection, Marker, Control, Two Tailed Test, Immunofluorescence

Journal: bioRxiv

Article Title: The R2TP chaperone assembles cellular machineries in intestinal CBC stem cells and progenitors

doi: 10.1101/2019.12.19.882712

Figure Lengend Snippet: (A) The graph depicts transcript levels of R2TP components in human primary colorectal tumor samples (n = 380; COADREAD cohort), as compared to normal solid tissues (n = 51). y-axis: Log2 normalized counts for the indicated transcript. Distributions are presented as box-and-whisker plots (center line: median; box limits, first and third quartiles; whiskers, 10th and 90th percentiles). Statistical significance was determined by one-way ANOVA (asterisk: P < 0.001). (B) Kaplan–Meier analysis of disease-free survival among 177 CRC patients according to the proportion of RPAP3-expressing cells in tumor tissues (C, P = 0.037). Solid green line and dashed blue line indicate High and Low proportion of RPAP3-expressing tumoral cells, respectively. Right panels show examples of CRC tissues with low (top) or high (bottom) RPAP3 expression. (C) Proposed model for R2TP activity in the small and large intestine. R2TP assembles cellular machineries such as RNA polymerases, snoRNPs, snRNPs and PIKKs-complexes in CBCs and progenitors in the proliferative compartment (blue cells). Differentiated cells (including Paneth cells from the small intestine crypts, in pink) mostly rely on the complexes assembled during the proliferative phase. A defect in R2TP activity induces client dysfunction, cell cycle arrest and apoptosis via p53, and eventually, epithelium degradation.

Article Snippet: Primary antibodies from Cell Signaling: ATM D2E2 (CST 2873S) rabbit at 1/1000, ATR E1S3S (CST 13934S) rabbit at 1/1000, mTOR (CST 2972S) rabbit 1/1000, p53 1C12 (CST2524S) mouse 1/1000; PRP8 (sc-30207, Santa Cruz) rabbit 1/200; from ABCAM: EFTUD2 (ab72456) rabbit 1/2000, GAPDH (ab8245 6C5) mouse 1/10000; and from Sigma: NOP58 (HPA018472) rabbit 1/100, RPAP3 (SAB1411438) rabbit 1/1000, Tubulin (I2G10) mouse 1/500, TRRAP 2TRR-1B3 (MABE1008) mouse 1/1000.

Techniques: Whisker Assay, Expressing, Activity Assay

Journal: Nucleic Acids Research

Article Title: CryoEM of RUVBL1–RUVBL2–ZNHIT2, a complex that interacts with pre-mRNA-processing-splicing factor 8

doi: 10.1093/nar/gkab1267

Figure Lengend Snippet: Purification and assembly of RUVBL1–RUVBL2–ZNHIT2ΔC. ( A ) Left top panels, domain architecture of the components of R2TP (TPRs, tetratricopeptide repeats; PBD, PIH1D1 binding domain; RBD, RUVBL2 binding domain; PIH, protein interacting with HSP90 domain; CS, CHORD-containig proteins and SGT1 domain). Domains involved in interactions within R2TP are indicated as links with dashed lines. Left bottom panel, constructs of human ZNHIT2 used in this work (H, HIT domain). Right panel, cartoon of the structure of the R2TP complex. ( B ) Diagram of the network of interactions described in this work for R2TP, ZNHIT2, ECD, AAR2 and PRPF8. ( C ) SDS-PAGE of purified ZNHIT2ΔC, GST-ZNHIT2ΔC, GST-ZNHIT2-C and GST-ZNHIT2-N. ( D ) GST-pull-down experiments to test the interaction of GST-ZNHIT2ΔC, GST-ZNHIT2-C and GST-ZNHIT2-N with RUVBL1, RUVBL2 and RUVBL1–RUVBL2 complexes (RUVBL1/2 for simplicity in figure legends hereafter). RUVBL1/2-ΔDII corresponds to mutants lacking part of the DII domain. RUVBL1/2-WB corresponds to mutants in the Walker B domains, able to bind ATP but affected in ATP hydrolysis. Control lanes for these experiments are shown in . ( E ) GST-pull-down experiment to evaluate the effect of the presence or absence of the nucleotides ATP, ADP or AMP–PNP in the interaction between RUVBL1–RUVBL2 and ZNHIT2ΔC. Control lanes for these experiments are shown in . ( F ) Cartoon showing the crosslinks found in RUVBL1–RUVBL2–ZNHIT2 complex detected by XL-MS.

Article Snippet: Primary antibodies used in western blotting with dilutions were as follows: monoclonal Anti-FLAG M2 antibody (Sigma Aldrich, F1804, 1:2000), PRPF8 (Abcam, ab87433; 1:300), EFTUD2 (Abcam, ab72456, 1:200), ZNHIT2 (Abcam, ab126133; 1:500), RUVBL1 (Cell signaling #12300; 1:500), RUVBL2 (Cell signaling #8959; 1:500), PIH1D1 (Invitrogen #PA5-61482, 1:1000), RPAP3 (Invitrogen #PA5-58334D; 1:500).

Techniques: Purification, Binding Assay, Construct, SDS Page, Control, Structural Proteomics

Journal: Nucleic Acids Research

Article Title: CryoEM of RUVBL1–RUVBL2–ZNHIT2, a complex that interacts with pre-mRNA-processing-splicing factor 8

doi: 10.1093/nar/gkab1267

Figure Lengend Snippet: CryoEM of the RUVBL1–RUVBL2-ZNHIT2ΔC complex. ( A ) SDS-PAGE of the purified RUVBL1–RUVBL2–ZNHIT2ΔC complex used for structural studies. ( B ) Representative 2D averages obtained from the cryoEM images of RUVBL1–RUVBL2- ZNHIT2ΔC. Grey arrowheads label some of the RUVBL1–RUVBL2 DII domains, while yellow arrowheads indicate the position of ZNHIT2ΔC. ( C ) Two views of the consensus cryoEM map of the RUVBL1–RUVBL2-ZNHIT2ΔC complex at 4.4 Å resolution. Density for ZNHIT2ΔC (yellow color) locates at the DII-face of the ring (grey color). ( D ) A view of one of the cryoEM maps obtained after classification using cryoDRGN , showing the DII-side of the RUVBL1–RUVBL2 ring. The density for ZNHIT2ΔC complex is divided in two lobules that correlate with the two domains predicted for ZNHIT2ΔC by AlphaFold . ( E ) Left panel, a tilted view of the cryoEM map in D showing the interaction of the large ZNHIT2ΔC domain. Both the atomic model of the RUVBL1–RUVBL2 complex and the ZNHIT2ΔC C-terminal domain are fitted within the map. Linked residues, identified by XL-MS, are shown as green dots. Each RUVBL subunit is labelled from 1 to 3 (subscript). Right panel, close-up view to highlight the proximity between the cross-linked residues in ZNHIT2 and the adjacent DII domains in the RUVBL1–RUVBL2 model.

Article Snippet: Primary antibodies used in western blotting with dilutions were as follows: monoclonal Anti-FLAG M2 antibody (Sigma Aldrich, F1804, 1:2000), PRPF8 (Abcam, ab87433; 1:300), EFTUD2 (Abcam, ab72456, 1:200), ZNHIT2 (Abcam, ab126133; 1:500), RUVBL1 (Cell signaling #12300; 1:500), RUVBL2 (Cell signaling #8959; 1:500), PIH1D1 (Invitrogen #PA5-61482, 1:1000), RPAP3 (Invitrogen #PA5-58334D; 1:500).

Techniques: SDS Page, Purification, Structural Proteomics

Journal: Nucleic Acids Research

Article Title: CryoEM of RUVBL1–RUVBL2–ZNHIT2, a complex that interacts with pre-mRNA-processing-splicing factor 8

doi: 10.1093/nar/gkab1267

Figure Lengend Snippet: ZNHIT2ΔC alters the conformation and ATPase activity of RUVBL2. ( A ) CryoEM density of the AAA-ring in RUVBL1–RUVBL2 after being processed independently of the flexible density for ZNHIT2ΔC and the DII domains. Density for nucleotides is only present in the RUVBL1 subunits (green density with ADP fitted inside). ADP molecules, without density accounting for them, are fitted in the described nucleotide binding sites of RUVBL2 only to highlight the lack of density in the region where nucleotide is present in human RUVBL1–RUVBL2 crystal structures (PDB ID 2XSZ). ( B ) Right panel, crystal structure of RUVBL1–RUVBL2 (PDB ID 2XSZ). Grey and pink arrowheads indicate the N-terminal regions of RUVBL1 and RUVBL2 respectively. These were absent in the atomic structure of RUVBL1–RUVBL2–ZNHIT2ΔC (indicated as pink empty arrowheads). Nucleotides are shown in green colour. ( C ) Close-up of nucleotide-binding sites for every RUVBL subunit after the interaction with ZNHIT2ΔC. Nucleotide molecules are show in green and nucleotide densities are displayed in semi-transparent green density. In RUVBL2, density for nucleotide is not observed, and this is indicated by placing ADP in the binding-site. ( D ) ATPase activity of RUVBL1–RUVBL2, wild type and mutants (RUVBL1 WB and RUVBL2 WB stand for the mutants whose Walker B motif has been altered to impair ATPase activity), in the presence and absence of ZNHIT2ΔC using the value of RUVBL1–RUVBL2 alone as 100%. Significance value of the paired Student's T-Test is shown for the RUVBL1–RUVBL2–ZNHIT2ΔC complex in comparison with the RUVBL1–RUVBL2 complex alone.

Article Snippet: Primary antibodies used in western blotting with dilutions were as follows: monoclonal Anti-FLAG M2 antibody (Sigma Aldrich, F1804, 1:2000), PRPF8 (Abcam, ab87433; 1:300), EFTUD2 (Abcam, ab72456, 1:200), ZNHIT2 (Abcam, ab126133; 1:500), RUVBL1 (Cell signaling #12300; 1:500), RUVBL2 (Cell signaling #8959; 1:500), PIH1D1 (Invitrogen #PA5-61482, 1:1000), RPAP3 (Invitrogen #PA5-58334D; 1:500).

Techniques: Activity Assay, Binding Assay, Comparison

Journal: Nucleic Acids Research

Article Title: CryoEM of RUVBL1–RUVBL2–ZNHIT2, a complex that interacts with pre-mRNA-processing-splicing factor 8

doi: 10.1093/nar/gkab1267

Figure Lengend Snippet: RUVBL1–RUVBL2 interacts with AAR2, ECD and ZNHIT2ΔC. ( A ) Pull-down experiments using the GST tag in ZNHIT2ΔC showing that ZNHIT2ΔC interacts with RUVBL1–RUVBL2 and ECD. ZNHIT2ΔC contaminants (*) coelute with AAR2 so the interaction between ZNHIT2ΔC and AAR2 could not be determined in this experiment. Electrophoretic protein mobility is shown with coloured arrowheads according to the protein labels. Main results are highlighted within rectangles. ( B ) Pull-down experiments using the Strep II tag in ECD confirmed its interaction with ZNHIT2ΔC and RUVBL1–RUVBL2. Electrophoretic protein mobility is shown with coloured arrowheads according to the protein labels. Main results are highlighted within rectangles. ( C ) Pull-downs using the His-tag in AAR2 confirmed its interaction with ECD and ZNHIT2ΔC. Albeit the His-tag been removed from RUVBL1–RUVBL2 some unspecific interaction was detected in the RUVBL1–RUVBL2 control lane (**). Electrophoretic protein mobility is shown with coloured arrowheads according to the protein labels. Main results are highlighted within rectangles. ( D ) Pull-down experiments using the Strep II tag in AAR2 probed that AAR2 interacts with RUVBL1–RUVBL2 both in the absence and in the presence of ZNHIT2ΔC. Main results are highlighted within rectangles.

Article Snippet: Primary antibodies used in western blotting with dilutions were as follows: monoclonal Anti-FLAG M2 antibody (Sigma Aldrich, F1804, 1:2000), PRPF8 (Abcam, ab87433; 1:300), EFTUD2 (Abcam, ab72456, 1:200), ZNHIT2 (Abcam, ab126133; 1:500), RUVBL1 (Cell signaling #12300; 1:500), RUVBL2 (Cell signaling #8959; 1:500), PIH1D1 (Invitrogen #PA5-61482, 1:1000), RPAP3 (Invitrogen #PA5-58334D; 1:500).

Techniques: Control

Journal: Nucleic Acids Research

Article Title: CryoEM of RUVBL1–RUVBL2–ZNHIT2, a complex that interacts with pre-mRNA-processing-splicing factor 8

doi: 10.1093/nar/gkab1267

Figure Lengend Snippet: Interactions between PRPF8 and RUVBL1–RUVBL2, RPAP3-PIH1D1, AAR2, ECD and ZNHIT2 ( A ) Flag-pull-down experiments showed a direct interaction between PRPF8 (bait) and RUVBL1–RUVBL2 (prey), which is not affected by the presence of ATP. Asterisks indicate contaminants that co-eluted with PRPF8. ( B ) Upper panel, pull-down experiments using Flag-PRPF8 (bait) and RPAP3-PIH1D1 in the presence or absence of RUVBL1–RUVBL2 were analyzed by western blot. Stained SDS-PAGE gels can be found in . Lower panel, pull-down experiment using the GST tag in RPAP3 within the RPAP3-PIH1D1 complex, analyzed by western blot. MW, molecular weight markers. ( C ) In the presence of ECD, ZNHIT2ΔC, RUVBL1–RUVBL2, RPAP3-PIH1D1 and AAR2 (preys), PRPF8 (bait) interacted with RUVBL1–RUVBL2, RPAP3-PIH1D1, AAR2 and ECD as show in the Flag-pull-down assay. Control experiments using a cell extract lacking the overexpressed PRPF8, but overexpressing the Flag-tag were run in parallel SDS-PAGE for the experiments and they are shown in . Stained SDS-PAGE gels can be found in .The presence of PRPF8, RUVBL1–RUVBL2, ECD, AAR2, RPAP3, PIH1D1 and ZNHIT2 in the Flag-pull-down assays was analyzed by western blot.

Article Snippet: Primary antibodies used in western blotting with dilutions were as follows: monoclonal Anti-FLAG M2 antibody (Sigma Aldrich, F1804, 1:2000), PRPF8 (Abcam, ab87433; 1:300), EFTUD2 (Abcam, ab72456, 1:200), ZNHIT2 (Abcam, ab126133; 1:500), RUVBL1 (Cell signaling #12300; 1:500), RUVBL2 (Cell signaling #8959; 1:500), PIH1D1 (Invitrogen #PA5-61482, 1:1000), RPAP3 (Invitrogen #PA5-58334D; 1:500).

Techniques: Western Blot, Staining, SDS Page, Molecular Weight, Pull Down Assay, Control, FLAG-tag