ANALYSIS OF RPB5 BINDING PROTEIN (RMP/URI) MEDIATED TRANSCRIPTIONAL REPRESSION IN PROSTATE CELLS.

Unconventional prefoldin RPB5 Interactor (URI) was identified as a protein that binds the RPB5 subunit of polymerases and functions as a transcriptional repressor (Dorjsuren N., MCB, 1998). Although URI interacts with RNA polymerases, our microarray analysis of genes affected by depletion of URI did not show a general effect on transcription and only 5% of the genes were affected. We also showed that URI binds and regulates the stability of Art-27, an epithelial-specific androgen receptor (AR) cofactor. URI, in complex with Art-27, represses androgen receptor mediated transcription and plays a critical role in anti-androgen action (Mita P., MCB, 2011). Although several proteins have been identified as URI interactors the mechanism by which URI represses transcription is unknown. To gain functional insight into the mechanism of URI repression we performed a mass spectrometry analysis of proteins interacting with URI in the nucleus of prostate cells. This analysis confirmed the previously reported interaction of URI with the three RNA polymerases, TFIIF, the Paf-1 complex and the R2TP/prefoldin-like complex. The analysis also identified novel interactors including the transcriptional repressor KAP1/TRIM28 and the phosphatase PP2A. We show that URI binds PP2A phosphatase and thereby regulates KAP1 phosphorylation. Depletion of URI induced KAP1 hyper-phosphorylation and de-repression of KAP1 regulated genes. These data are interesting in light of the recently discovered role of KAP1 in retroelement repression. Moreover, our mass spectrometry analysis showed a correlation between the URI phosphorylation state, polII CTD phosphorylation, and the recruitment of proteins involved in active polII transcription supporting the idea that URI phosphorylation could function as a metabolically activated “switch” that controls polII elongation on specific genes. We also show that URI is involved in the DNA damage response in mammalian cells and that the URI/Art-27 complex plays a role in prostate cell differentiation and luminal-epithelial cell specification. Collectively, the data presented in this thesis demonstrate that URI, together with its “partner” Art-27, is part of a novel transcriptional repressor complex that plays a role in a wide range of cellular processes including cell differentiation, DNA damage and tumorigenesis.