Interaction with general transcription factor IIF (TFIIF) is required for the suppression of activated transcription by RPB5-mediating protein (RMP)

Abstract

RMP was reported to regulate transcription via competing with HBx to bind the general transcription factor IIB (TFIIB) and interacting with RPB5 subunit of RNA polymerase II as a corepressor of transcription regulator. However, our present research uncovered that RMP also regulates the transcription through interaction with the general transcription factors IIF (TFIIF), which assemble in the preinitiation complex and function in both transcription initiation and elongation. With in vitro pull-down assay and Far-Western analysis, we demonstrated that RMP could bind with bacterially expressed recombinant RAP30 and RAP74 of TFIIF subunits. In the immunoprecipitation assay in COS1 cells cotransfected with FLAG-tagged RMP or its mutants, GST-fused RAP30 and RAP74 were co-immunoprecipitated with RMP in approximately equal molar ratio, which suggests that RAP30 and RAP74 interact with RMP as a TFIIF complex. Interestingly both RAP30 and RAP74 interact with the same domain (D5) of the C-terminal RMP of 118-aminoacid residuals which overlaps with its TFIIB-binding domain. Internal deletion of D5 region of RMP abolished its binding ability with both subunits of TFIIF, while D5 domain alone was sufficient to interact with TFIIF subunits. The result of luciferase assay showed that overexpression of RMP, but not the mutant RMP lacking D5 region, suppressed the transcription activated by Gal-VP16, suggesting that interaction with TFIIF is required for RMP to suppress the activated transcription. The interaction between RMP and TFIIF may be an additional passway for RMP to regulate the transcription, or alternatively TFIIF may cooperate with RPB5 and TFIIB for the corepressor function of RMP.

Regulation of Androgen Receptor-Mediated Transcription by RPB5 Binding Protein URI/RMP

“…Our focus, however, was on the role of URI as a transcription regulator. Consistent with the idea that URI is also present in the nucleus, our mass spectrometry analysis of URI nuclear interactors identified several nuclear proteins, such as RPB1 phos- (30,33) strengthens the idea that nuclear URI plays an important role in transcription regulation independent of its cytoplasmic role as a chaperone for Pol II complex assembly. The costabilization of URI and Art-27 makes it very difficult to discern independent functions for the two proteins.…”

“…It also binds other nuclear proteins involved in transcription, including the general transcription factor IIF (TFIIF) (15,30) and components of the Paf-1 complex that promote polymerase II (pol II) phosphorylation and histone modifications during elongation (33). Although URI was initially demonstrated to function as a transcriptional repressor (8), its role in transcriptional regulation is not well understood.…”

New insights into the biogenesis of nuclear RNA polymerases?This paper is one of a selection of papers published in this special issue entitled “Canadian Society of Biochemistry, Molecular & Cellular Biology 52nd Annual Meeting — Protein Folding: Principles and Diseases” and has undergone the Journal's usual peer review process.

“…This activity would proceed through a competition for binding of the RPB5 subunit (Nikolov and Burley 1997;Dorjsuren et al 1998;Wei et al 2003), consistent with the proposed role of RPB5 on activated transcription (Miyao and Woychik 1998).…”

URI Regulates KAP1 Phosphorylation and Transcriptional Repression via PP2A Phosphatase in Prostate Cancer Cells

“…In the nucleus, URI functions as a transcriptional repressor that binds RPB5, the shared subunit of the RNA polymerases (5). Despite several reports demonstrating that URI binds assembling (3) and transcribing (6) RNA polymerase II, and key regulators of RNA polymerase II transcription (7,8) and in conjunction with prefoldin-like protein ART-27, represses androgen receptor-dependent gene expression (9) in prostate cells, little is known about the mechanism of URI transcriptional repression. In Saccharomyces cerevisiae, URI binds the phosphorylated C-terminal domain (CTD) 2 of RNA polymerase II and affects the recruitment of the chromatin remodeling complex RSC (10).…”