Table 1.
URI is part of the R2TP/prefoldin-like complex.
Figure 1.
URI binds RPB5 protein and affects its protein stability and gene transcription.
a) 293 cells were transfected with RPB5 and URI wild type (URI WT) or URI deleted of the RPB5 binding domain (URIΔRPB5). 48 hrs after transfection cells were lysed and part of the lysate used for the immunoprecipitation of URI using FLAG antibodies. Cells were either growth in the absence or presence of 25 µM MG132. The expression and immunoprecipiation of URI and RPB5 was analyzed by Western blotting. Tubulin was used as loading control. b) mRNA was isolated from 293 stable cell lines overexpressing an empty vector (293-vector) or a construct encoding FLAG-URI (293-URI). URI and RPB5 mRNA were quantified by qPCR. The URI and RPB5 mRNA of 293-vector cells were set as 1. c) The indicated µg of pcDNA3-FLAG-URI construct were transfected in LNCaP cells. 48 hours after transfection proteins and mRNA were isolated. RPB5 and URI mRNA and protein expression were measured by qPCR (left panels) and Western blot (right panels). RPB5 protein expression (quantified by densitometry analysis of the reported Western blot) is also plotted with the µg of transfected URI (R2 is 0.729). d) mRNA and proteins were isolated from LNCaP cells treated with control siRNA (siCtrl) or siRNA directed against URI (siURI). RPB5 mRNA and URI and RPB5 proteins were measured by qPCR (bottom) and Western blot (top). e) In vitro transcribed/translated and 35S labelled RPB5, URI WT and URIΔRPB5 proteins were mixed and used to immunoprecipitate URI with a FLAG antibody. Input solutions and immunoprecipitated complexes were analyzed by SDS-PAGE.
Figure 2.
RPB5 binding is not necessary for URI mediated repression of AR mediated transcription.
LB1-LNCaP cells were transfected with an empty vector, a vector encoding URI or URIΔRPB5. 0.5 µg or 1 µg of URIΔRPB5 construct were transfected. After 24 hrs of hormone starvation cells were treated with or without 10 nM R1881 for an additional 24 hrs. The luciferase units normalized by protein content are reported.
Figure 3.
a) LNCaP-vector cells and LNCaP-URI stable cell lines were used to immunoprecipitate URI, FLAG-URI and Art-27. The immunocomplexes were analyzed by Western blotting using the indicated antibodies. ERK1 is used as loading control. LNCaP cells transiently overexpressing an empty vector or a construct encoding PDRG1 were also analyzed for the expression of PDRG1, URI and Art-27. URI and PDRG1 mRNA expression in control LNCaP cells (vector) and LNCaP cells overexpressing URI (URI) treated with or without synthetic androgen R1881 are reported on the right panels. b) URI was depleted from LNCaP cells by sequential siRNA transfection as described in materials and methods. After knock down cells were treated with or without 10 nM R1881 for 24 hrs and then lysed in Triton buffer or their mRNA was isolated. Cell lysates were analysed for Art-27, PDRG1 and URI expression by Western blotting and qPCR (right panel). ERK1 protein is used as loading control and LNCaP cell lysates from normally cycling cells cultured in complete media are used for comparison. The numbers reported below the PDRG1 blotting are densitometry analysis of PDRG1 protein bands (ImageJ).
Figure 4.
URI binds PDRG1 through the prefoldin-like domain.
a) Schematic of the seven URI constructs used to map the domains of interaction between URI and PDRG1. The known URI domains are reported and the numbers on top indicate the corresponding amino-acid number. b) An empty vector (vect.), PDRG1 (gray box; 5 µg) and the different URI constructs were transfected into HEK293 cells (10 µg total DNA transfected). URI deleted of the prefoldin domain is labeled PFD. 48 hrs after transfection cells were lysed. Part of the lysate was used as INPUT (left panel) and another part (≈1 mg of proteins) was used to immunoprecipitate URI using FLAG antibodies. The immunocomplexes were analyzed by Western blot using the indicated antibodies. Arrowheads indicate the URI deletions/truncations.
Figure 5.
URI depletion or overexpression does not alter RUVBL1 and RUVBL2 protein levels.
a) Cytoplasmic (C) and nuclear extracts (N) were isolated from LNCaP cells stably overexpressing URI. Nuclear extracts were used to immunoprecipitate URI and co-immunoprecipiatin of URI with RUVBL1 and RUVBL2 was analyzed by Western blot analysis. The white arrowhead indicates the RUVBL2 band while the arrows indicates a nonspecific band recognized by the RUVBL2 antibody. LNCaP cell lines stably expressing a non silencing shRNA (shNS), a shRNA against URI (shURI), an empty vector (vect.) or a construct encoding URI (URI) were lysed and the expression of RUVBL1 and RUVBL2 protein and mRNA was analyzed by Western blot analysis (b) and qPCR (c). hsp90 and ERK1 were used as loading controls.
Figure 6.
URI shuttling between the cytoplasm and the nucleus is dependent on CRM1/XPO1-exportin.
LNCaP cells stably overexpressing an URI-EGFP fusion protein were used to visualize URI localization (a,b and c). Cells were treated with α-amanitin (d,e and f), α-amanitin + leptomycin B (g, h and i) or with actinomycin D (j–l). Cells were counterstained with DAPI (visualized as blue fluorescence). Single colors and merged pictures are presented.
Figure 7.
URI phosphorylation on T241, S243 and URI acetylation on K89 does not interfere with URI interaction with RPB5 and Art-27.
293 cells were transfected with a construct encoding RPB5 (a) or HA-Art-27 (b) and a construct encoding URI wild type (WT), URI T241A/S243A (URI T_S)(a) or URI K89A (b). FLAG-URI was then immunoprecipitated with FLAG antibodies and HA-Art-27 or RPB5 were analyzed by Western blotting using specific antibodies. Tubulin was used as loading control.
Table 2.
Identified sites of potential URI post-transcriptional modification.
Figure 8.
Model for the interaction of the R2TP/prefoldin-like components.
The prefoldin-like components of the complex (URI, Art-27, PDRG1 and presumably PFD2 and PFD6) interact through the “hook” of their prefoldin-like domain. URI also binds RPB5 with a.a. 177–224 which most likely connects the whole prefoldin-like complex to the RNA polymerase II.