URI binds RPB5 protein and affects its protein stability and gene transcription.

<p>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.