Journal: Nature Communications

Article Title: RPAP3 provides a flexible scaffold for coupling HSP90 to the human R2TP co-chaperone complex

doi: 10.1038/s41467-018-03942-1

Figure Lengend Snippet: Mapping the interactions in human R2TP core components. a A cartoon for sequence and domains of the components of the human R2TP complex. b GST pull-down experiments depicting the interactions between the several regions in RPAP3 and PIH1D1. FL stands for full length, CS for the CS domain in PIH1D1, and MW for molecular weight markers. Be aware that for simplification, several PIH1D1 and RPAP3 constructs are indicated within the same lines on top of the gel. Some minor contaminants are present in some of the samples. c Pull-down experiments showing that removal of residues 401–420 from an RPAP3 construct eliminates the interaction with the CS domain in PIH1D1. d Pull-down experiments demonstrating the interaction of RPAP3–RBD with RUVBL2. This interaction is not affected when the DII domains in RUVBL2 are removed

Article Snippet: The RPAP3 full length (FL) was purchased from GenScript.

Techniques: Sequencing, Molecular Weight, Construct

Journal: Nature Communications

Article Title: RPAP3 provides a flexible scaffold for coupling HSP90 to the human R2TP co-chaperone complex

doi: 10.1038/s41467-018-03942-1

Figure Lengend Snippet: Cryo-EM imaging of RUVBL1–RUVBL2 and the RBD domain. a 2D averages corresponding to top and side views obtained from cryo-EM images of the RUVBL1–RUVBL2 preparation in an ADP-containing buffer. b After incubation with RPAP3 430–665 , RBDs decorate the ATPase side of both RUVBL rings without disrupting the dodecamer, and a representative 2D average of the complex between RUVBL1–RUVBL2 and RBD is shown. At the right end of the panels, one view of the 3D structure of RUVBL1–RUVBL2–RBD complex with RBD domains in yellow. Note that one of the RBD domains in the bottom ring is less visible at the threshold used for rendering, probably reflecting variable occupancy. Also, the scale of the 3D structure has been enlarged with respect to the 2D average, for clarity

Article Snippet: The RPAP3 full length (FL) was purchased from GenScript.

Techniques: Cryo-EM Sample Prep, Imaging, Incubation

Journal: Nature Communications

Article Title: RPAP3 provides a flexible scaffold for coupling HSP90 to the human R2TP co-chaperone complex

doi: 10.1038/s41467-018-03942-1

Figure Lengend Snippet: Cryo-EM imaging of the R2TP complex. a Pull-down experiments showing the in vitro reconstitution of R2TP. M indicates molecular weight markers. b Purification of RUVBL1–RUVBL2 and PIH1D1–RPAP3 sub-complexes, used for the reconstitution of R2TP for cryo-EM. M indicates molecular weight markers. c Two representative side view averages of R2TP. RUVBL1–RUVBL2 rings are decorated by the RBD at the top (labeled with white arrows). A blurred and very flexible region locates at the bottom of the ring. d A representative side view average of R2TP reconstructed using the RPAP3–ΔNT–PIH1D1 sub-complex and RUVBL1–RUVBL2. Flexible regions at the bottom end of R2TP disappear when the N-terminal half of RPAP3 is removed, but dodecameric RUVBL1–RUVBL2 is disrupted. e 3D structure of R2TP obtained applying 3-fold symmetry. RBDs are bound to RUVBL1–RUVBL2 but the flexible regions in the complex are not resolved. Scale bar, 2.5 nm

Article Snippet: The RPAP3 full length (FL) was purchased from GenScript.

Techniques: Cryo-EM Sample Prep, Imaging, In Vitro, Molecular Weight, Purification, Labeling

Journal: Nature Communications

Article Title: RPAP3 provides a flexible scaffold for coupling HSP90 to the human R2TP co-chaperone complex

doi: 10.1038/s41467-018-03942-1

Figure Lengend Snippet: A cartoon for the structural and functional model for R2TP. a Human R2TP. HSP90 dimers can engage with each R2TP complex with sufficient conformational flexibility to reach and act in a diversity of client proteins. Up to 3 RBDs serve to anchor 3 RPAP3 to the RUVBL1–RUVBL2 scaffold, whereas a central segment of RPAP3 helps to recruit PIH1D1. The number of RPAP3 molecules per RUVBL ring in vivo is not known, and two options are shown in the figure. A long and poorly structured link between the RBD and TPR domains in RPAP3 results in substantially conformational flexibility of the TPR regions. For simplicity, although 3 RBDs are bound to the RUVBL ring, only 2 RPAP3s are shown bound to HSP90 in the cartoon. b Yeast R2TP. Conformational adaptability of yeast R2TP is limited to the flexibility of the C-terminal tails in Hsp90. Only one Hsp90 binds each R2TP.

Article Snippet: The RPAP3 full length (FL) was purchased from GenScript.

Techniques: Functional Assay, In Vivo

Journal: Nature Communications

Article Title: RPAP3 provides a flexible scaffold for coupling HSP90 to the human R2TP co-chaperone complex

doi: 10.1038/s41467-018-03942-1

Figure Lengend Snippet: Mapping the interactions in human R2TP core components. a A cartoon for sequence and domains of the components of the human R2TP complex. b GST pull-down experiments depicting the interactions between the several regions in RPAP3 and PIH1D1. FL stands for full length, CS for the CS domain in PIH1D1, and MW for molecular weight markers. Be aware that for simplification, several PIH1D1 and RPAP3 constructs are indicated within the same lines on top of the gel. Some minor contaminants are present in some of the samples. c Pull-down experiments showing that removal of residues 401–420 from an RPAP3 construct eliminates the interaction with the CS domain in PIH1D1. d Pull-down experiments demonstrating the interaction of RPAP3–RBD with RUVBL2. This interaction is not affected when the DII domains in RUVBL2 are removed

Article Snippet: The RPAP3 1-430 , RPAP3 1-400 , RPA3 1-420 , RPAP3 395-665 , RPAP3 430-665 , RPAP3 523-665 , RPAP3 430-541 , RPAP3 541-665 , and RPAP3 395-665 (RPAP3-ΔNT) gene truncations were cloned using NdeI sites and ligation-free cloning using infusion cloning (Clonetech lab Inc.) into a modified pGex6p modified plasmid named p3E (University of Sussex, UK), which resulted in N-terminal GST-tagged RPAP3 genes (Supplementary Table ).

Techniques: Sequencing, Molecular Weight, Construct

Journal: Nature Communications

Article Title: RPAP3 provides a flexible scaffold for coupling HSP90 to the human R2TP co-chaperone complex

doi: 10.1038/s41467-018-03942-1

Figure Lengend Snippet: Cryo-EM imaging of RUVBL1–RUVBL2 and the RBD domain. a 2D averages corresponding to top and side views obtained from cryo-EM images of the RUVBL1–RUVBL2 preparation in an ADP-containing buffer. b After incubation with RPAP3 430–665 , RBDs decorate the ATPase side of both RUVBL rings without disrupting the dodecamer, and a representative 2D average of the complex between RUVBL1–RUVBL2 and RBD is shown. At the right end of the panels, one view of the 3D structure of RUVBL1–RUVBL2–RBD complex with RBD domains in yellow. Note that one of the RBD domains in the bottom ring is less visible at the threshold used for rendering, probably reflecting variable occupancy. Also, the scale of the 3D structure has been enlarged with respect to the 2D average, for clarity

Article Snippet: The RPAP3 1-430 , RPAP3 1-400 , RPA3 1-420 , RPAP3 395-665 , RPAP3 430-665 , RPAP3 523-665 , RPAP3 430-541 , RPAP3 541-665 , and RPAP3 395-665 (RPAP3-ΔNT) gene truncations were cloned using NdeI sites and ligation-free cloning using infusion cloning (Clonetech lab Inc.) into a modified pGex6p modified plasmid named p3E (University of Sussex, UK), which resulted in N-terminal GST-tagged RPAP3 genes (Supplementary Table ).

Techniques: Cryo-EM Sample Prep, Imaging, Incubation

Journal: Nature Communications

Article Title: RPAP3 provides a flexible scaffold for coupling HSP90 to the human R2TP co-chaperone complex

doi: 10.1038/s41467-018-03942-1

Figure Lengend Snippet: Cryo-EM imaging of the R2TP complex. a Pull-down experiments showing the in vitro reconstitution of R2TP. M indicates molecular weight markers. b Purification of RUVBL1–RUVBL2 and PIH1D1–RPAP3 sub-complexes, used for the reconstitution of R2TP for cryo-EM. M indicates molecular weight markers. c Two representative side view averages of R2TP. RUVBL1–RUVBL2 rings are decorated by the RBD at the top (labeled with white arrows). A blurred and very flexible region locates at the bottom of the ring. d A representative side view average of R2TP reconstructed using the RPAP3–ΔNT–PIH1D1 sub-complex and RUVBL1–RUVBL2. Flexible regions at the bottom end of R2TP disappear when the N-terminal half of RPAP3 is removed, but dodecameric RUVBL1–RUVBL2 is disrupted. e 3D structure of R2TP obtained applying 3-fold symmetry. RBDs are bound to RUVBL1–RUVBL2 but the flexible regions in the complex are not resolved. Scale bar, 2.5 nm

Article Snippet: The RPAP3 1-430 , RPAP3 1-400 , RPA3 1-420 , RPAP3 395-665 , RPAP3 430-665 , RPAP3 523-665 , RPAP3 430-541 , RPAP3 541-665 , and RPAP3 395-665 (RPAP3-ΔNT) gene truncations were cloned using NdeI sites and ligation-free cloning using infusion cloning (Clonetech lab Inc.) into a modified pGex6p modified plasmid named p3E (University of Sussex, UK), which resulted in N-terminal GST-tagged RPAP3 genes (Supplementary Table ).

Techniques: Cryo-EM Sample Prep, Imaging, In Vitro, Molecular Weight, Purification, Labeling

Journal: Nature Communications

Article Title: RPAP3 provides a flexible scaffold for coupling HSP90 to the human R2TP co-chaperone complex

doi: 10.1038/s41467-018-03942-1

Figure Lengend Snippet: A cartoon for the structural and functional model for R2TP. a Human R2TP. HSP90 dimers can engage with each R2TP complex with sufficient conformational flexibility to reach and act in a diversity of client proteins. Up to 3 RBDs serve to anchor 3 RPAP3 to the RUVBL1–RUVBL2 scaffold, whereas a central segment of RPAP3 helps to recruit PIH1D1. The number of RPAP3 molecules per RUVBL ring in vivo is not known, and two options are shown in the figure. A long and poorly structured link between the RBD and TPR domains in RPAP3 results in substantially conformational flexibility of the TPR regions. For simplicity, although 3 RBDs are bound to the RUVBL ring, only 2 RPAP3s are shown bound to HSP90 in the cartoon. b Yeast R2TP. Conformational adaptability of yeast R2TP is limited to the flexibility of the C-terminal tails in Hsp90. Only one Hsp90 binds each R2TP.

Article Snippet: The RPAP3 1-430 , RPAP3 1-400 , RPA3 1-420 , RPAP3 395-665 , RPAP3 430-665 , RPAP3 523-665 , RPAP3 430-541 , RPAP3 541-665 , and RPAP3 395-665 (RPAP3-ΔNT) gene truncations were cloned using NdeI sites and ligation-free cloning using infusion cloning (Clonetech lab Inc.) into a modified pGex6p modified plasmid named p3E (University of Sussex, UK), which resulted in N-terminal GST-tagged RPAP3 genes (Supplementary Table ).

Techniques: Functional Assay, In Vivo

Journal: Nature Communications

Article Title: The RPAP3-Cterminal domain identifies R2TP-like quaternary chaperones

doi: 10.1038/s41467-018-04431-1

Figure Lengend Snippet: Solution structure of the C-terminal domain of human RPAP3 a Schematic representation of the human R2TP complex. b Domain architecture of RPAP3 (numbering corresponds to amino-acids of isoform 1). c Conservation of RPAP3 and PIH repertoires across Eukaryotes. Members in which TPR (Pfam: 13414) or Dynein attachment (Pfam: 15867) domains are associated to the canonical RPAP3_C are colored as indicated at the left. Clades or species that have lost flagella or in which cilia are not motile are in gray background. Members that were not found are indicated by x. d Backbone view of the superposition of the best 20 NMR structures of human RPAP3-Cter, with α-helices indicated in violet. e Sequence of RPAP3-Cter, with corresponding α-helices. f Backbone orthogonal views (180 °C) of RPAP3-Cter structure in ribbon representation with corresponding α-helices

Article Snippet: Recombinant 13 C/ 15 N-labeled RPAP3 535-665 domain with a cleavable 6xHis-tag were overexpressed in E. coli (DE3) pRARE2 (Novagen) overnight at 20 °C in a minimal M9 medium complemented with 15 N-NH 4 Cl and 13 C-d6-Glucose and purified on TALON beads (Clonteth) in 25 mM HEPES, pH 7.5, 300 mM NaCl and eluted from the resin by cleavage with the HRV-3C protease (GE Healthcare).

Techniques: Sequencing

Journal: Nature Communications

Article Title: The RPAP3-Cterminal domain identifies R2TP-like quaternary chaperones

doi: 10.1038/s41467-018-04431-1

Figure Lengend Snippet: NMR and refinement statistics for top-20 RPAP3 535-665 structures

Article Snippet: Recombinant 13 C/ 15 N-labeled RPAP3 535-665 domain with a cleavable 6xHis-tag were overexpressed in E. coli (DE3) pRARE2 (Novagen) overnight at 20 °C in a minimal M9 medium complemented with 15 N-NH 4 Cl and 13 C-d6-Glucose and purified on TALON beads (Clonteth) in 25 mM HEPES, pH 7.5, 300 mM NaCl and eluted from the resin by cleavage with the HRV-3C protease (GE Healthcare).

Techniques:

Journal: Nature Communications

Article Title: The RPAP3-Cterminal domain identifies R2TP-like quaternary chaperones

doi: 10.1038/s41467-018-04431-1

Figure Lengend Snippet: RPAP3-Cter interacts with RUVBL1/2 hexamers. a SILAC proteomic analysis of RPAP3-Cter. The graph depicts the proteins identified in anti-GFP immuno-precipitates of HeLa cells expressing GFP-RPAP3-Cter. Each dot is a protein and the color code is indicated below the graph. X -axis: protein abundance (Log10 of signal intensity); y-axis: enrichment over a control IP (Log2 of SILAC ratio). b NMR interaction analysis of RPAP3-Cter with recombinant RUVBL1/2 complex. The graph depicts 1D NMR METHYL-SOFAST-HMQC spectra in the methyl region of 13 C-labeled RPAP3-Cter alone (top lane) or mixed with recombinant RUVBL1/2 complex (bottom lane). Intensity of the NMR signal (arbitrary units, Y -axis) is plotted against the 1 H chemical shift (in ppm, X -axis). c SPR binding assays of RPAP3-Cter with RUVBL1/2. The graph depicts the response upon injecting the RUVBL1/2 complex (t = 0 s), or upon washing (t = 300 s), on immobilized RPAP3-Cter. X -axis: time (s); Y -axis: response (arbitrary units). These data have been obtained with the same batch of RUVBL1/2 complex as in the control experiment (Fig. S2E, F). d Chromatographic analysis of the RUVBL complexes. The graph depicts the chromatograms of purified RUVBL1–RUVBL2 (dashed gray, left Y axis) or RUVBL1-RUVBL2-RPAP3-Cter (black line, right Y axis), on a Superose 6 16/70 XK. X-axis: elution volume; Y-axis: absorbance. e Electrophoresis of the purified RUVBL1–RUVBL2–RPAP3–Cter complex. The gel shows the peak fraction of the complex eluted from the column (black line in d), with a purity estimated to ~95 %. Lane 1: Precision Plus Protein Unstained Standards (Biorad); Lane 2: denatured RUVBL1–RUVBL2–RPAP3–Cter complex. Black and white arrows: RUVBL1 and RUVBL2 (52 and 53 kDa, respectively); gray arrow: RPAP3-Cter (15 kDa). f Native mass spectrometry analysis of recombinant RUVBL complexes. The upper mass spectrum presents the purified RUVBL1/2 complex. The bottom mass spectrum presents the same complex after addition of RPAP3-Cter. Y-axis: signal intensity; X-axis: m/z. Insets: zoom over the 8000–9000 m/z region. Schematics depict the complex observed. Blue: RUVBL proteins; red: RPAP3-Cter

Article Snippet: Recombinant 13 C/ 15 N-labeled RPAP3 535-665 domain with a cleavable 6xHis-tag were overexpressed in E. coli (DE3) pRARE2 (Novagen) overnight at 20 °C in a minimal M9 medium complemented with 15 N-NH 4 Cl and 13 C-d6-Glucose and purified on TALON beads (Clonteth) in 25 mM HEPES, pH 7.5, 300 mM NaCl and eluted from the resin by cleavage with the HRV-3C protease (GE Healthcare).

Techniques: Expressing, Recombinant, Labeling, Binding Assay, Purification, Electrophoresis, Mass Spectrometry

Journal: Nature Communications

Article Title: The RPAP3-Cterminal domain identifies R2TP-like quaternary chaperones

doi: 10.1038/s41467-018-04431-1

Figure Lengend Snippet: RUVBL1/2:RPAP3–Cter affinity and kinetic interaction parameters determined by SPR

Article Snippet: Recombinant 13 C/ 15 N-labeled RPAP3 535-665 domain with a cleavable 6xHis-tag were overexpressed in E. coli (DE3) pRARE2 (Novagen) overnight at 20 °C in a minimal M9 medium complemented with 15 N-NH 4 Cl and 13 C-d6-Glucose and purified on TALON beads (Clonteth) in 25 mM HEPES, pH 7.5, 300 mM NaCl and eluted from the resin by cleavage with the HRV-3C protease (GE Healthcare).

Techniques:

Journal: Nature Communications

Article Title: The RPAP3-Cterminal domain identifies R2TP-like quaternary chaperones

doi: 10.1038/s41467-018-04431-1

Figure Lengend Snippet: The RPAP3 C-terminal domain interacts with R2TP clients via RUVBL1/2 multimers. a Yeast two-hybrid analysis of interactions between RUVBL2 and RPAP3-Cter mutants. Alix is a negative control. ***: strong interaction; **: medium; *: weak; −: no interaction. b Molecular surface representation of RPAP3-Cter structure by specifying the location of the mutants that lost interaction with RUVBL1/2. c LUMIER assay showing the in vivo interaction between RPAP3-Cter and RUVBL1/2 mutant proteins. Top panel: schematic representation of the assay. Bottom panel: graph plotting the IP efficiency of the indicated proteins. The values are the IP efficiencies of the co-precipitation of the RL fusion proteins (IP/Input), normalized by the IP/Input values obtained with the anti-FLAG IP of the 3xFLAG-FFL fusion protein. Error bars: standard deviation. Stars: values significantly greater than six-times the mean value obtained in the control IPs without anti-FLAG antibody (Ct). ** p -value < 0.001 (Z-test). d,e SILAC proteomic analysis of the partners of RPAP3-Cter-Mut1 and RPAP3-Cter-Mut2, respectively. Legend as in Fig.

Article Snippet: Recombinant 13 C/ 15 N-labeled RPAP3 535-665 domain with a cleavable 6xHis-tag were overexpressed in E. coli (DE3) pRARE2 (Novagen) overnight at 20 °C in a minimal M9 medium complemented with 15 N-NH 4 Cl and 13 C-d6-Glucose and purified on TALON beads (Clonteth) in 25 mM HEPES, pH 7.5, 300 mM NaCl and eluted from the resin by cleavage with the HRV-3C protease (GE Healthcare).

Techniques: Negative Control, In Vivo, Mutagenesis, Standard Deviation

Journal: Nature Communications

Article Title: The RPAP3-Cterminal domain identifies R2TP-like quaternary chaperones

doi: 10.1038/s41467-018-04431-1

Figure Lengend Snippet: RPAP3-like and PIH1-like proteins interact with each other. a Architecture of the human proteins containing a RPAP3-Cterminal domain (RPAP3-C), or a PIH domain (PIH). Coiled-coil (CC), CHORD-containing proteins and SGT1 domain (CS) and TPR domains (TPR) are also indicated. Different splicing isoforms of RPAP3 and DYX1C1 are shown, with their variable domains in hatched violet (RPAP3), or yellow (DYX1C1). b Summary of pairwise LUMIER interaction assays between the indicated proteins. The values are the efficiencies of the co-precipitation of the RL fusion proteins (IP/Input), expressed in percent of the efficiencies obtained with the 3xFLAG-FFL fusions. p -values are shown in Supplementary Fig. . c LUMIER interaction assays between RL-RUVBL1/2 and RPAP3-like proteins tagged with 3xFLAG-FL. Legend as in Fig. . Stars: values significantly greater than six-times the mean value obtained in the control IP (Ct). ** p -value < 0.001 ( Z -test)

Article Snippet: Recombinant 13 C/ 15 N-labeled RPAP3 535-665 domain with a cleavable 6xHis-tag were overexpressed in E. coli (DE3) pRARE2 (Novagen) overnight at 20 °C in a minimal M9 medium complemented with 15 N-NH 4 Cl and 13 C-d6-Glucose and purified on TALON beads (Clonteth) in 25 mM HEPES, pH 7.5, 300 mM NaCl and eluted from the resin by cleavage with the HRV-3C protease (GE Healthcare).

Techniques:

Journal: Cancer Science

Article Title: Heat shock protein 90 regulates phosphatidylinositol 3‐kinase‐related protein kinase family proteins together with the RUVBL1/2 and Tel2‐containing co‐factor complex

doi: 10.1111/j.1349-7006.2011.02112.x

Figure Lengend Snippet: Heat shock protein 90 (Hsp90) interacts with two other phosphatidylinositol 3‐kinase‐related protein kinase (PIKK) regulators, RUVBL1/2 and Tel2 and their associated proteins. (A) RUVBL1 interacted with Hsp90, a Hsp90 co‐factor (NOP17), URI complex and Tel2 complex. Tet‐inducible streptavidin‐binding peptide (SBP)‐tagged RUVBL1 stable HEK 293 cells or control cells, which express tag peptides only, were treated with 1 ng/mL doxycycline for 3 days. Cytoplasmic cell extracts were affinity purified with streptavidin sepharose, and biotin‐eluted fractions were analyzed by western blotting with the indicated antibodies. (B,C) Protein interactions of Tel2, SMG‐10, Tti2 and URI. HeLa TetOff cells were immunoprecipitated with anti‐Tti2, anti‐Tel2 antiserum or normal rabbit serum (NRS) (B), or anti‐SMG‐10, URI, or normal rabbit IgG (NRIgG) (C). The immunoprecipitates were analyzed by western blotting with the indicated antibodies. Input: 1, 0.33, 0.11 and 0.037% (B) or 0.5, 0.17 and 0.06% (C) of the amount immunoprecipitated. (D) RPAP3 interacts with all PIKK. HeLa TetOff cells were transfected with pcDNA5/FRT/TO/NTAP‐GST, pcDNA5/FRT/TO/NTAP‐NOP17, pcDNA5/FRT/TO/NTAP‐RPAP3 or pcDNA5/FRT/TO/NTAP‐SMG‐10. The cell extracts were subjected to affinity purification with streptavidin sepharose 36 h later and analyzed by western blotting with indicated antibodies. ATM, ataxia telangiectasia mutated; ATR, ATM‐and Rad3‐related; DNA‐PKcs, DNA‐dependent protein kinase catalytic subunit; mTOR, mammalian target of rapamycin; NOP17, nucleolar protein 17; RPB5, RNA polymerase II subunit 5; SMG‐1, suppressor with morphological effect on genitalia 1; TRRAP, transformation/transcription domain‐associated protein.

Article Snippet: The following siRNA target sequences were used: RUVBL1, siGENOME duplex D‐008977‐02 (Dharmacon, Lafayette, CO, USA); RUVBL2, siGENOME duplex D‐012299‐03 (Dharmacon); SMG‐10, siGENOME duplex J‐014188‐05 (Dharmacon); Tti2, Hs_Tti2_2 HP Validated siRNA SI00401660 (Qiagen, Valencia, CA, USA); Tel2, Hs_KIAA0683_4 HP Validated siRNA SI00454909 (Qiagen); URI, Hs_C19orf2_4 HP Validated siRNA SI00322462 (Qiagen); RPB5, Hs_POLR2E_3 HP Validated siRNA SI00689073 (Qiagen); NOP17, Hs_FLJ20643_4 HP Validated siRNA SI00120148 (Qiagen); RPAP3, Hs_FLJ21908_3 HP Validated siRNA SI00399875 (Qiagen); DNA‐PKcs, #1: Hs_PRKDC_ 8_HP Validated siRNA SI02663633 (Qiagen), #2: Hs_ PRKDC_2_HP Validated siRNA SI00093079 (Qiagen); ATM, #1: Hs_ATM_4_HP Validated siRNA SI00000847 (Qiagen), #2: Hs_ATM_14_HP Validated siRNA SI03068506 (Qiagen); ATR, #1: Hs_ATR_12_HP Validated siRNA SI02664347 (Qiagen), #2: Hs_ATR_2_HP Validated siRNA SI00023107 (Qiagen); mTOR, #1: Hs_FRAP1_7 _HP Validated siRNA SI03023587 (Qiagen), #2: Hs_FRAP1_8 _HP Validated siRNA SI03064985 (Qiagen); SMG‐1, #1: Mm_2610207I05Rik_4 HP Validated siRNA SI02765546 (Qiagen), #2: GTGTATGTGCGCCAAAGTA; TRRAP, #1: Hs_TRRAP_2_HP Validated siRNA SI00052591 (Qiagen), #2: Hs_TRRAP_3_HP Validated siRNA SI00052598 (Qiagen); and NS siRNA, All Star Negative Control siRNA (Qiagen).

Techniques: Binding Assay, Control, Affinity Purification, Western Blot, Immunoprecipitation, Transfection, Transformation 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: ZNHIT2 forms a complex with R2TP in vitro and in cells. ( A ) Consecutive pull-down experiments to analyze the formation of R2TP-ZNHIT2ΔC complexes. His-RUVBL1 was used in a first pulldown step, and then the eluted material was the input of a second pulldown experiment using the GST tag in ZNHIT2ΔC. ( B ) Volcano plot comparing interactors of RPAP3 M626A/F630R versus RPAP3-WT is shown. Two-sample Student's t -test was performed. Only interactors with a P -value <0.05 and a log 2 ratio ≤ 2 were considered downregulated in the mutant condition. The permutation-based FDR was estimated to be below 5%. ( C ) The 22 interactors significantly down-represented in RPAP3 M626A/F630R versus RPAP3-WT are indicated in the table. Those linked with U5 snRNP or its maturation are in bold. ( D ) Immunoprecipitation (IP) of transiently transfected HEK293T cells with RPAP3-WT RPAP3 M626A/F630R . Anti-FLAG-IPs, whole-cell extracts (WCE, 1%) and flow through (FT) were subjected to western analysis using the indicated antibodies. Blots are representative of 5 independent experiments. Data (mean ± SEM of five independent experiments) were normalized by total FLAG and expressed as fold-change of the different proteins indicated association with FLAG with respect to the RPAP3-WT condition. Statistical significance was analyzed using unpaired t -test. ** P < 0.01, *** P < 0.001.

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: In Vitro, Mutagenesis, Immunoprecipitation, Transfection, Western Blot

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, FLAG-tag

Journal: Nature Communications

Article Title: RPAP3 provides a flexible scaffold for coupling HSP90 to the human R2TP co-chaperone complex

doi: 10.1038/s41467-018-03942-1

Figure Lengend Snippet: Mapping the interactions in human R2TP core components. a A cartoon for sequence and domains of the components of the human R2TP complex. b GST pull-down experiments depicting the interactions between the several regions in RPAP3 and PIH1D1. FL stands for full length, CS for the CS domain in PIH1D1, and MW for molecular weight markers. Be aware that for simplification, several PIH1D1 and RPAP3 constructs are indicated within the same lines on top of the gel. Some minor contaminants are present in some of the samples. c Pull-down experiments showing that removal of residues 401–420 from an RPAP3 construct eliminates the interaction with the CS domain in PIH1D1. d Pull-down experiments demonstrating the interaction of RPAP3–RBD with RUVBL2. This interaction is not affected when the DII domains in RUVBL2 are removed

Article Snippet: The RPAP3 1-430 , RPAP3 1-400 , RPA3 1-420 , RPAP3 395-665 , RPAP3 430-665 , RPAP3 523-665 , RPAP3 430-541 , RPAP3 541-665 , and RPAP3 395-665 (RPAP3-ΔNT) gene truncations were cloned using NdeI sites and ligation-free cloning using infusion cloning (Clonetech lab Inc.) into a modified pGex6p modified plasmid named p3E (University of Sussex, UK), which resulted in N-terminal GST-tagged RPAP3 genes (Supplementary Table ).

Techniques: Sequencing, Molecular Weight, Construct

Journal: Nature Communications

Article Title: RPAP3 provides a flexible scaffold for coupling HSP90 to the human R2TP co-chaperone complex

doi: 10.1038/s41467-018-03942-1

Figure Lengend Snippet: Cryo-EM imaging of RUVBL1–RUVBL2 and the RBD domain. a 2D averages corresponding to top and side views obtained from cryo-EM images of the RUVBL1–RUVBL2 preparation in an ADP-containing buffer. b After incubation with RPAP3 430–665 , RBDs decorate the ATPase side of both RUVBL rings without disrupting the dodecamer, and a representative 2D average of the complex between RUVBL1–RUVBL2 and RBD is shown. At the right end of the panels, one view of the 3D structure of RUVBL1–RUVBL2–RBD complex with RBD domains in yellow. Note that one of the RBD domains in the bottom ring is less visible at the threshold used for rendering, probably reflecting variable occupancy. Also, the scale of the 3D structure has been enlarged with respect to the 2D average, for clarity

Article Snippet: The RPAP3 1-430 , RPAP3 1-400 , RPA3 1-420 , RPAP3 395-665 , RPAP3 430-665 , RPAP3 523-665 , RPAP3 430-541 , RPAP3 541-665 , and RPAP3 395-665 (RPAP3-ΔNT) gene truncations were cloned using NdeI sites and ligation-free cloning using infusion cloning (Clonetech lab Inc.) into a modified pGex6p modified plasmid named p3E (University of Sussex, UK), which resulted in N-terminal GST-tagged RPAP3 genes (Supplementary Table ).

Techniques: Cryo-EM Sample Prep, Imaging, Incubation

Journal: Nature Communications

Article Title: RPAP3 provides a flexible scaffold for coupling HSP90 to the human R2TP co-chaperone complex

doi: 10.1038/s41467-018-03942-1

Figure Lengend Snippet: Cryo-EM imaging of the R2TP complex. a Pull-down experiments showing the in vitro reconstitution of R2TP. M indicates molecular weight markers. b Purification of RUVBL1–RUVBL2 and PIH1D1–RPAP3 sub-complexes, used for the reconstitution of R2TP for cryo-EM. M indicates molecular weight markers. c Two representative side view averages of R2TP. RUVBL1–RUVBL2 rings are decorated by the RBD at the top (labeled with white arrows). A blurred and very flexible region locates at the bottom of the ring. d A representative side view average of R2TP reconstructed using the RPAP3–ΔNT–PIH1D1 sub-complex and RUVBL1–RUVBL2. Flexible regions at the bottom end of R2TP disappear when the N-terminal half of RPAP3 is removed, but dodecameric RUVBL1–RUVBL2 is disrupted. e 3D structure of R2TP obtained applying 3-fold symmetry. RBDs are bound to RUVBL1–RUVBL2 but the flexible regions in the complex are not resolved. Scale bar, 2.5 nm

Article Snippet: The RPAP3 1-430 , RPAP3 1-400 , RPA3 1-420 , RPAP3 395-665 , RPAP3 430-665 , RPAP3 523-665 , RPAP3 430-541 , RPAP3 541-665 , and RPAP3 395-665 (RPAP3-ΔNT) gene truncations were cloned using NdeI sites and ligation-free cloning using infusion cloning (Clonetech lab Inc.) into a modified pGex6p modified plasmid named p3E (University of Sussex, UK), which resulted in N-terminal GST-tagged RPAP3 genes (Supplementary Table ).

Techniques: Cryo-EM Sample Prep, Imaging, In Vitro, Molecular Weight, Purification, Labeling

Journal: Nature Communications

Article Title: RPAP3 provides a flexible scaffold for coupling HSP90 to the human R2TP co-chaperone complex

doi: 10.1038/s41467-018-03942-1

Figure Lengend Snippet: A cartoon for the structural and functional model for R2TP. a Human R2TP. HSP90 dimers can engage with each R2TP complex with sufficient conformational flexibility to reach and act in a diversity of client proteins. Up to 3 RBDs serve to anchor 3 RPAP3 to the RUVBL1–RUVBL2 scaffold, whereas a central segment of RPAP3 helps to recruit PIH1D1. The number of RPAP3 molecules per RUVBL ring in vivo is not known, and two options are shown in the figure. A long and poorly structured link between the RBD and TPR domains in RPAP3 results in substantially conformational flexibility of the TPR regions. For simplicity, although 3 RBDs are bound to the RUVBL ring, only 2 RPAP3s are shown bound to HSP90 in the cartoon. b Yeast R2TP. Conformational adaptability of yeast R2TP is limited to the flexibility of the C-terminal tails in Hsp90. Only one Hsp90 binds each R2TP.

Article Snippet: The RPAP3 1-430 , RPAP3 1-400 , RPA3 1-420 , RPAP3 395-665 , RPAP3 430-665 , RPAP3 523-665 , RPAP3 430-541 , RPAP3 541-665 , and RPAP3 395-665 (RPAP3-ΔNT) gene truncations were cloned using NdeI sites and ligation-free cloning using infusion cloning (Clonetech lab Inc.) into a modified pGex6p modified plasmid named p3E (University of Sussex, UK), which resulted in N-terminal GST-tagged RPAP3 genes (Supplementary Table ).

Techniques: Functional Assay, In Vivo

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 Schematic representation of R2TP with its four subunits (RPAP3, PIH1D1, and the RUVBL1/2 heterohexamer). RPAP3 is the core subunit that contacts directly HSP90, PIH1D1 and RUVBL1/2. b β galactosidase activity in Rpap3 wtsi/+ small intestines (top) and colon (bottom), as compared to negative controls ( n = 2). Scale bars = 50 μm are identical for all images. c IHC of Pih1d1 in the small intestine. Counter coloration of DNA with hematoxylin, with magnification (bottom). Top picture: bar represents 50 μm. Inset: arrows point to CBC stem cells, intercalated between Paneth cells with distinctive granules in the cytoplasm; bar is 20 μm. Micrograph is representative of n = 3. d Depletion of Rpap3 after tamoxifen injection. Western blots were revealed with antibodies against the indicated proteins in extracts of the jejunum (left) and the colonic (right) epitheliums of RPAP3 flox/flox controls (blue) or VilCreER T2 ; RPAP3 flox/flox animals (red), 5 days after the first tamoxifen injection. Each lane was loaded with the lysate obtained from a single animal and were verified for n = 12 small intestines and 8 colons, in three independent experiments. Molecular sizes are indicated on the right. e Individual weight variations in females (top panel) and males (bottom panel) of tamoxifen treated VilCreER T2 ; RPAP3 flox/flox animals (red curves, n = 6 females and n = 5 males) and RPAP3 flox/flox controls (blue curves, n = 8 females and n = 6 males). Individual weights were set at 100% for each animal at day 0, and analyzed by two-way ANOVA (genotype affects weights with p < 0.0001 in male and females) and Bonferroni’s post hoc multiple comparison tests (** p < 0.01; *** p < 0.001; **** p < 0.0001—see ). f Length of small intestines and colons from VilCreER T2 ; RPAP3 flox/flox mice (red points) and controls (blue points) measured at day 8 and day 10 of females (top, n = 3) and males (bottom, n = 4) were analyzed by two-tailed unpaired t test with Welch’s correction (females: * p = 0.0177, t = 4.750, df = 3; males: *** p = 0.0001, t = 14.14, df = 4). Statistics data represent individual assays with mean ± S.E.M. g Representative images of small intestine (white arrowheads) from VilCreER T2 ; RPAP3 flox/flox animals, which were filled with liquid (left panel) or blood (right panel) from day 8 to 10; a control organ is shown above. 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: Activity Assay, Injection, Western Blot, Comparison, Two Tailed Test, Control

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 Schematic representation of the experimental setting: 8-week-old mice of the indicated genotype received two sequential injections of tamoxifen 24 h apart, and were analyzed 5 to 8 days after the first injection. 2 h before each sacrifice, BrdU was injected intraperitoneally to detect cells in S-phase (thin arrows). b 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). c Representative pictures of jejunum tissue sections stained with HE (top) or by IHC with anti-Ki67 antibody (bottom, Ki67 signal is brown) at indicated days after the first tamoxifen injection. Pink arrows point towards Paneth cells and black arrows towards CBC stem cells. Scale bar is shown in control HE panel. Each panel is representative of 8 to 12 animals analyzed in three independent experiments. Scale bar is identical for all panels and is 15 μm. d Representative pictures of jejunum taken from control (boxed in blue) and VilCreER T2 ; Rpap3 flox/flox animals (boxed in red), stained by IHC with anti-BrdU antibodies (brown arrows). n = 3–6 animals/ time point from two independent experiments. Scale bar is shown in control panel and is 50 μm. e Graph shows mean number of BrdU + cells/crypt in controls and VilCreER T2 ; Rpap3 flox/flox animals at day 7. Each point represents the average number of BrdU + cells calculated in n > 35 crypts from two different zones per animal ( n = 3). Mean values with S.E.M are indicated for each experimental group. Unpaired two-tailed t test with Welch’s corrections ( t = 11.66, df = 2) indicates significant difference between control and VilCreER T2 ; Rpap3 flox/flox animals ( p = 0.0073; n = 3). 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: Injection, Staining, Control, Two Tailed Test

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 , b Staining for Olfm4 in the jejunum from control (top panel) and VilCreER T2 ; Rpap3 flox/flox animals 7 days ( a ) or 6 to 8 days after the first tamoxifen injection. Panels are representative for 2 to 4 animals/time point from at least two independent experiments. Scale bar is 20 μm in ( a ) and 50 μm in ( b ). 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 Representative micrographs of tissue sections immuno-stained for lysozyme, a specific marker of Paneth cells, in the jejunum of control (top) and VilCreER T2 ; Rpap3 flox/flox mice from day 6 to day 8. Panels are representative for 2 to 3 animals/time point from two independent experiments. Scale bar, identical in all pictures, is 50 μm. e Total number of apoptotic cells identified by cleaved caspase 3 (cleaved cas3 + ) per surface (mm 2 ) of jejunum for each mouse analyzed, at day 6. 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 cleaved caspase 3 in the jejunum. In control animals, cleaved caspase 3 + cells (brown arrows) are mainly detected at the tip of the villi, as a result of epithelial 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. Scale bar, identical in all pictures, is 50 μm. 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, Control, Injection, Marker, Two Tailed Test

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 Micrographs are tissue sections stained for p53 by immunofluorescence in controls (top) and VilCreER T2 ; Rpap3 flox/flox mice (bottom) at day 6, representative of n = 7 animals from three independent experiments. Nuclei were stained with DAPI. Scale bar is 50 μm and is identical for all pictures. b Micrographs are tissue sections stained by immunofluorescence for p53 (Cy5, red) and lysozyme (Alexa 488, green), a marker of Paneth cells, in VilCreER T2 ; Rpap3 flox/flox mice at day 6 ( n = 4). Nuclei were stained with DAPI. Scale bar, 50 μm, is identical for all pictures. c Pictures of jejunum sections stained with HE (top) or by IHC with anti-Ki67 antibodies (bottom) in P53 KO ( VilCreER T2 ; Rpap3 flox/+ ; Trp53 flox/flox ), Rpap3 KO (VilCreER T2 ; Rpap3 flox/flox ; Trp53 flox/+ ) and double Rpap3 KO; P53 KO (VilCreER T2 ; Rpap3 flox/flox ; Trp53 flox/flox ) mice at day 6 to 8 after the first tamoxifen injection. Pictures are representative for each single KO ( n = 3) and double KO ( n = 5–7) mice, from three independent experiments. Scale bar, 50 μm, is identical for all pictures. d Pictures of jejunum sections stained by IHC with anti-Rpb1 antibody in control, P53 KO ( VilCreER T2 ; Rpap3 flox/+ ; Trp53 flox/flox ), Rpap3 KO (VilCreER T2 ; Rpap3 flox/flox ; Trp53 flox/+ ) and double Rpap3 KO; P53 KO (VilCreER T2 ; Rpap3 flox/flox ; Trp53 flox/flox ) mice at day 6 following tamoxifen injection. Pictures are representative of n = 3 for each single KO, n = 7 for double KO, from two different experiments. Scale bar, 50 μm, is identical for all pictures. e Total number of apoptotic cells at day 6 per surface (mm 2 ) of the jejunum for each mouse analyzed. n = 2 for wild type, n = 3 for each single KO, n = 7 for double KO, from two different experiments. Mean values with S.E.M are indicated for experimental groups with n > 3. One-way ANOVA analysis ( p = 0.0006) with Bonferroni’s multiple comparison post-test (** p < 0.001). 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, Immunofluorescence, Marker, Injection, Control, Comparison

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: 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 Schematic representation of the experimental setting. Eight-week-old Lgr5-GFP-IRES-CreER T2 ; Rpap3 flox/flox mice received five sequential intra-peritoneal injections of tamoxifen and were analyzed 7 or 10 days after the first injection. In this genetic model, the Cre is expressed in the Lgr5 + CBC stem cells labeled by GFP (see scheme on the right). b Representative images of tissue sections labeled by immunofluorescence with antibodies against GFP (green) and Rpb1 (red), with DAPI counter-staining of nuclei (blue). Please note the mosaic expression of GFP. Panels are representative for 5 animals/time point from two independent experiments. White arrow: GFP + crypts. Asterisks: GFP − crypts. Scale bars (40 or 20 μm) are identical for matching panels. c Images are intestine tissue sections of wild-type animals stained for BrdU. Animals received one BrdU injection and were sacrificed at the indicated time point. The experiment was repeated twice (2 to 4 animals/time point from two different experiments). Scale bars (50 μm) are identical for all pictures.

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: Injection, Labeling, Immunofluorescence, Staining, Expressing

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 The graph depicts transcript levels of R2TP components in human primary colorectal tumor samples ( n = 380), as compared to normal solid tissues ( n = 51) from COADREAD cohort. 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 (* 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. Solid green line and dashed blue line indicate high and low proportion of RPAP3-expressing tumoral cells, respectively. Statistical significance was determined by log-rank test ( P = 0.037). Right panels show examples of CRC tissues with low (top) or high (bottom) RPAP3 expression, with scale bar. 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: 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: Whisker Assay, Expressing, Activity Assay

Journal: Molecular cell

Article Title: Crosstalk between RNA Pol II C-Terminal Domain Acetylation and Phosphorylation via RPRD Proteins

doi: 10.1016/j.molcel.2019.04.008

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies RPRD1B (Rabbit) Bethyl AB_11218400 RPRD1B (Mouse) Novus AB_2285416 RRPD1A Santa Cruz sc514724 Pol II (8WG16) Abcam AB_306327 Pol II (4H8) Abcam AB_304868 Pol II (H224) Santa Cruz AB_2268548 Pol II (K7ac) This Paper N/A RPAP3 Bethyl AB_2621049 HA (Rat) Sigma AB_10094468 HA Agarose Bead Sigma AB_257974 HA Magnetic Bead Sigma AB_2749815 Normal IgG (Rabbit) Santa Cruz AB_737197 Normal IgG (Mouse) Santa Cruz AB_737182 HSP90 Abcam AB_444729 HDAC1 Abcam AB_2264059 AffiniPure Goat Anti-Mouse HRP Jackson Immunoresearch AB_10015289 AffiniPure Goat Anti-Rabbit HRP Jackson Immunoresearch AB_2313567 AffiniPure Goat Anti-Rat HRP Jackson Immunoresearch AB_2338128 Chemicals, Peptides, and Recombinant Proteins Panobinostat Santa Cruz CAS 404950-80-7 Nicotinamide Sigma CAS 98-92-0 α-Amanitin Santa Cruz CAS 23109-05-9 CTD Peptides Peptide 2.0 N/A Critical Commercial Assays QIAquick PCR Purification Kit Qiagen 28104 RNeasy Plus Mini Kit Qiagen 74136 Ovation Ultralow System V2 NuGEN 0344-32 Deposited Data RNA Pol II ChIP-seq Data GEO GSE124996 RNA Seq Data GEO GSE125486 Western Blotting Data Mendeley Data DOI: 10.17632/f6w4wzb9dr.1 Structural Modeling Mendeley Data DOI: 10.17632/f6w4wzb9dr.1 SILAC Data This Paper Table S1 Experimental Models: Cell Lines NIH3T3 ATCC CRL-1658 293T ATCC CRL-3216 Software and Algorithms Skewer v 0.1.124 Jiang et al., 2014 N/A Bowtie v 1.1.2 Langmead et al., 2009 N/A MACS2 v 2.1.0.20150731 Zhang et al., 2008 N/A DeepTools v 2.2.3 Ramirez et al., 2016 N/A MaxQuant v 1.2.5.8 Cox and Mann, 2008 N/A Open in a separate window KEY RESOURCES TABLE RPRD proteins interact with the RNA Pol II CTD in an acetylation-dependent manner RPRD CTD-interacting domain amide residues stabilize acetylated CTD heptads KDAC inhibition enahnces Pol II CTD K7-acetylation & reduces S5-phosphorylation RPRD1B knockdown disrupts Pol II CTD deacetylase & CTD phosphatase activity

Techniques: Recombinant, Purification, RNA Sequencing Assay, Western Blot, Software

Journal: Marine Drugs

Article Title: Target Identification of the Marine Natural Products Dictyoceratin-A and -C as Selective Growth Inhibitors in Cancer Cells Adapted to Hypoxic Environments

doi: 10.3390/md17030163

Figure Lengend Snippet: Binding of probe A ( 3 ) to the candidate proteins in cell lysates. Cell lysates (100 μg protein each), prepared from DU145 cells cultured under hypoxic conditions (H, lanes 2, 4, and 6) or normoxic conditions (N, lanes 1, 3, and 5) were incubated with probe A ( 3 ). The probe with bound protein was then separated from the supernatant by the addition of streptavidin-conjugated Dynabeads. The resulting supernatants and Dynabeads were used for western blotting analysis to detect RBM28, RPAP3, MIA3, EIF5AL1, and TRMT6.

Article Snippet: Polyclonal rabbit anti-RPAP3 antibodies were obtained from Bethyl Laboratories, Inc. (Montgomery, TX, USA).

Techniques: Binding Assay, Cell Culture, Incubation, Western Blot

Journal: Marine Drugs

Article Title: Target Identification of the Marine Natural Products Dictyoceratin-A and -C as Selective Growth Inhibitors in Cancer Cells Adapted to Hypoxic Environments

doi: 10.3390/md17030163

Figure Lengend Snippet: Growth of RBM28-, RPAP3-, and MIA30-knockdown DU145 cells under normoxic and hypoxic conditions. ( a ) The expression levels of the indicated proteins in each knockdown cell line were analyzed by western blotting. The expression level of each protein was calculated from its relative density, which was normalized to that of β-actin. ( b ) The proliferation rates of MIA3-, RBM28-, and RPAP3-knockdown DU145 cells were investigated under normoxic and hypoxic conditions. The growth inhibition rate was calculated as a percentage relative to the negative control cells. Differences were considered significant at * p < 0.01.

Article Snippet: Polyclonal rabbit anti-RPAP3 antibodies were obtained from Bethyl Laboratories, Inc. (Montgomery, TX, USA).

Techniques: Knockdown, Expressing, Western Blot, Inhibition, Negative Control

Journal: Marine Drugs

Article Title: Target Identification of the Marine Natural Products Dictyoceratin-A and -C as Selective Growth Inhibitors in Cancer Cells Adapted to Hypoxic Environments

doi: 10.3390/md17030163

Figure Lengend Snippet: Expression of hypoxia-related proteins in RPAP3-knockdown DU145 cells. The expression levels of the indicated hypoxia-related proteins in RPAP3-knockdown DU145 cells were compared with those of wild-type DU145 cells treated with/without dictyoceratin-A ( 1 ) or -C ( 2 ) (10 µM each) using western blotting analysis. The expression level of each protein was calculated from its relative density, which was normalized to that of β-actin.

Article Snippet: Polyclonal rabbit anti-RPAP3 antibodies were obtained from Bethyl Laboratories, Inc. (Montgomery, TX, USA).

Techniques: Expressing, Knockdown, Western Blot

Journal: Marine Drugs

Article Title: Target Identification of the Marine Natural Products Dictyoceratin-A and -C as Selective Growth Inhibitors in Cancer Cells Adapted to Hypoxic Environments

doi: 10.3390/md17030163

Figure Lengend Snippet: Effect of dictyoceratin-A ( 1 ) on RPAP3-overexpressing DU145 cells. ( a ) The expression levels of RPAP3 protein in stable RPAP3-overexpressing cells were compared with those in wild-type DU145 cells using western blotting. The expression levels of RPAP3 were calculated based on the relative density of bands, normalized to that of β-actin. ( b ) Antiproliferative activity of dictyoceratin-A ( 1 ) in wild-type and RPAP3-overexpressing DU145 cells. The growth inhibition rate was calculated as a percentage relative to that in control cells. Differences were considered significant at * p < 0.01.

Article Snippet: Polyclonal rabbit anti-RPAP3 antibodies were obtained from Bethyl Laboratories, Inc. (Montgomery, TX, USA).

Techniques: Expressing, Western Blot, Activity Assay, Inhibition, Control