Mechanism of Repression of RNA Polymerase I Transcription by the Retinoblastoma Protein

Abstract

The retinoblastoma susceptibility gene product pRb restricts cellular proliferation by affecting gene expression by all three classes of nuclear RNA polymerases. To elucidate the molecular mechanisms underlying pRb-mediated repression of ribosomal DNA (rDNA) transcription by RNA polymerase I, we have analyzed the effect of pRb in a reconstituted transcription system. We demonstrate that pRb, but not the related protein p107, acts as a transcriptional repressor by interfering with the assembly of transcription initiation complexes. The HMG box-containing transcription factor UBF is the main target for pRb-induced transcriptional repression. UBF and pRb form in vitro complexes involving the C-terminal part of pRb and HMG boxes 1 and 2 of UBF. We show that the interactions between UBF and TIF-IB and between UBF and RNA polymerase I, respectively, are not perturbed by pRb. However, the DNA binding activity of UBF to both synthetic cruciform DNA and the rDNA promoter is severely impaired in the presence of pRb. These studies reveal another mechanism by which pRb suppresses cell proliferation, namely, by direct inhibition of cellular rRNA synthesis.The cell division cycle proceeds in a temporally ordered series of events that involves both positive and negative regulators. Among the negative regulators are the product of the retinoblastoma susceptibility gene (RB-1), pRb, and the related p107 and p130 proteins (reviewed in references 9 and 43). It was recognized early that mutations that affect the retinoblastoma gene product are implicated in perturbed control of the cell cycle and the genesis of a wide variety of human tumors (20). pRb appears to act as a signal transducer connecting the cell cycle clock with the transcriptional machinery. Loss of pRb function deprives the cell of an important mechanism for inhibiting cell proliferation through modulation of gene expression. The growth-suppressing activity is related to the ability of pRb to block progression from the G 1 to the S phase. pRb is a phosphoprotein whose state of phosphorylation fluctuates through different phases of the cell cycle. Underphosphorylated pRb predominates in G 1 , and phosphorylation by the cyclin-dependent kinases seems to relieve the G 1 block and permit entry into the S phase (12, 16). Thus, pRb may act as a key switch in allowing cells to go through the cell cycle.One approach to deciphering the function of pRb was the identification of its interacting partners. pRb binds and controls the activity of various cellular and viral proteins, including the E2F family of transcription factors, Elf-1, MyoD, Pu.1, ATF-2, SP1, c-Abl, and some of the DNA tumor virus-transforming proteins, such as E1A protein, simian virus 40 large T antigen, and human papillomavirus E7 protein (reviewed in reference 41). Binding of the hypophosphorylated form of pRb to E2F proteins specifically prevents the expression of genes that are required for DNA replication (17).Previous studies examining the mechanism of growth suppression have focused on the ability of p...

Partially Processed pre-rRNA Is Preserved in Association with Processing Components in Nucleolus-derived Foci during Mitosis

“…An immediate supply of preribosomal particles ready to be made into ribosomes may provide efficiency to the daughter cells for rapid recovery from the dimunition of the synthetic machinery after mitosis. This could be especially important since the rate of pre-rRNA transcription appears to be at a low level during early G 1 phase and does not reach substantial rates until late G 1 phase (Voit et al, 1997). Work in this laboratory suggests that most of the nucleolar proteins and snoRNAs in the NDF reenter the reforming nucleoli at the end of mitosis (Dundr et al, 1996(Dundr et al, , 1997; this work), although we have not determined the fate of the pre-RNA molecules.…”

Human tumor suppressor p14ARF negatively regulates rRNA transcription and inhibits UBF1 transcription factor phosphorylation

“…Indeed, part of the nucleolar Arf has been shown to reside at the fibrillar/granular junction of the nucleolus (Weber et al, 1999), suggesting that Arf could shuttle between these two compartments to interact with other factors. A similar mechanism can be evoked to explain how the nucleoplasmic tumor suppressor pRb can directly interact with UBF in the fibrillar center of the nucleolus (Voit et al, 1997). To go further, co-immunoprecipitations were performed in proliferating cells expressing various levels of p14 ARF and cells undergoing cell cycle arrest.…”

“…All these kinases have been found overexpressed in many cancers. Several tumor suppressors have also been shown to directly interact with UBF: pRb or p130 inhibit in that way subsequent recruitment of cofactors required for rRNA transcription (Cavanaugh et al, 1995;Voit et al, 1997;Hannan et al, 2000a, b;Ciarmatori et al, 2001). p53 also interferes with Pol I activity by destabilizing the SL1-UBF complex required for transcriptional initiation on the rRNA promoter (Zhai and Comai, 2000).…”

The role of c-myc in cellular growth control

“…Cmyc directly interacts with speci®c domains on pRb in vitro and alters transcription of target genes (Hateboer et al, 1993;Maheswaran et al, 1991;Rustgi et al, 1991). Furthermore, pRb directly binds the UBF component of the PolI activation apparatus and inhibits PolI transcription in vitro (Cavanaugh et al, 1995;Voit et al, 1997). The combination of these two interactions might suggest a mechanism for c-Myc to regulate PolI transcription.…”