p53 is a major tumour suppressor that is inactivated in a large proportion of human cancers. We show that p53 serves as a general repressor of transcription by RNA polymerase (pol) III. It can inhibit the synthesis of a range of essential small cellular RNAs including tRNA, 5S rRNA and U6 snRNA, as well as viral products such as the adenovirus VAI RNA. Fibroblasts derived from p53 knock-out mice display a substantial increase in pol III transcriptional activity. Endogenous cellular p53 is shown to interact with the TATA-binding protein (TBP)-containing general factor TFIIIB, thereby compromising its function severely. However, assembly of TFIIIB into a pre-initiation complex confers substantial protection against the inhibitory effects of p53. Since TFIIIB is an essential determinant of the biosynthetic capacity of cells, its release from repression by p53 may contribute to a loss of growth control during the development of many tumours.

(White et al., 1996). When two human osteosarcoma lines were comThe tumour suppressor protein RB restricts cellular pared, RB-deficient SAOS2 cells were found to have a growth. This may involve inhibiting the synthesis of more active pol III transcription apparatus than RBtRNA and 5S rRNA by RNA polymerase (pol) III. We positive U2OS cells. In addition, primary fibroblasts from have shown previously that RB can repress pol III RB-knockout mice were shown to have much higher pol transcription when overexpressed either in vitro or III activity than primary fibroblasts from wild-type mice. in vivo. We also demonstrated that pol III activity isNo general increase in pol II activity was seen when elevated substantially in primary fibroblasts from SAOS2 cells were compared with U2OS cells or when RB-deficient mice. Here we address the molecular Rb -/-fibroblasts were compared with Rb ϩ/ϩ fibroblasts. mechanism of this regulation. RB is shown to repressThese experiments established that endogenous RB plays all types of pol III promoter. It can do this even if an important role in suppressing pol III transcription. added after transcription complex assembly. Func-The minimal region of RB that is required for growth tional assays demonstrate that RB targets specifically suppression comprises amino acid residues 379-928 (Qin the general pol III factor TFIIIB. A physical interaction et al., 1992). The same sequences are sufficient to inhibit between TFIIIB and RB is indicated by fractionation, pol III transcription (White et al., 1996). Various deletions pull-down and immunoprecipitation data. We show that remove segments from within this region abolish the that TFIIIB activity is elevated in primary fibroblasts ability of RB to regulate pol III (White et al., 1996). from RB-deficient mice. TFIIIB is a multisubunit Several naturally occurring mutations that prevent RB complex that includes the TATA-binding protein (TBP) from functioning as a tumour suppressor also inhibit its and a TFIIB-related factor called BRF. We show that ability to repress pol III (White et al., 1996). This raises RB itself contains regions of homology to both TBP the possibility that regulation of pol III contributes to and BRF and propose a model in which RB disrupts the tumour suppressive activity of RB (Nasmyth, 1996; TFIIIB by mimicking these two components.White, 1997). Keywords: RB/RNA polymerase III/TBP/TFIIIB/ The viral oncoproteins SV40 large T antigen and transcription adenovirus E1A can bind to RB and neutralize its function (Whyte, 1995). Both T antigen and E1A can also activate pol III transcription (Loeken et al., 1988; Patel and Jones, Introduction 1990;White et al., 1996). One way in which they may achieve this is by overcoming the repression of pol III The retinoblastoma protein (RB) is a 105 kDa nuclear transcription by RB (White et al., 1996). The ability of phosphoprotein that is encoded by an important tumour these oncoproteins to activate pol III by relieving the suppressor gene (reviewed by Weinberg, 1995;Whyte, physiolo...

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Increased rates of RNA polymerase (pol) III transcription constitute a central feature of the mitogenic response, but little is known about the mechanism(s) responsible. We demonstrate that the retinoblastoma protein RB plays a major role in suppressing pol III transcription in growth-arrested fibroblasts. RB knockout cells are compromised in their ability to down-regulate pol III following serum withdrawal. RB binds and represses the pol III-specific transcription factor TFIIIB during G 0 and early G 1 , but this interaction decreases as cells approach S phase. Full induction of pol III coincides with mid-to late G 1 phase, when RB becomes phosphorylated by cyclin D-and E-dependent kinases. TFIIIB only associates with the underphosphorylated form of RB, and overexpression of cyclins D and E stimulates pol III transcription in vivo. The RB-related protein p130 also contributes to the repression of TFIIIB in growth-arrested fibroblasts. These observations provide insight into the mechanisms responsible for controlling pol III transcription during the switch between growth and quiescence.The retinoblastoma protein RB is a highly abundant tumor suppressor that can bind and regulate a variety of transcription factors (reviewed in Refs. 1-4). One example that has been added recently to the growing list of RB-binding proteins is the RNA polymerase (pol) 1 III-specific factor TFIIIB (5, 6). Recombinant RB was shown to bind to TFIIIB in vitro and repress its activity (5, 6). Furthermore, coimmunoprecipitation and cofractionation experiments demonstrated a stable association between endogenous cellular RB and TFIIIB (6). The functional significance of this interaction was shown in studies of RB knockout mice, since primary fibroblasts from RbϪ/Ϫ mice display elevated TFIIIB activity relative to fibroblasts derived from their wild-type siblings (6). These results establish TFIIIB as a bona fide target for repression by RB. Similar approaches have shown that TFIIIB is also bound and repressed by the RB-related proteins p107 and p130 (7).TFIIIB is required for the expression of all pol III templates (reviewed in Refs. 8 and 9). It serves to recruit the polymerase to a promoter and position it over the transcription start site (10). By interacting with this general factor, RB appears able to regulate the expression of all pol III-transcribed genes, including tRNA, 5 S rRNA, U6 small nuclear RNA, VA1, and Alu genes (5, 6, 11). Since a high rate of tRNA and rRNA synthesis is required to sustain rapid growth, it has been speculated that the inhibition of pol III transcription may contribute to the growth suppression capacity of RB (12)(13)(14).RB function is regulated by cyclin-dependent kinases (reviewed in Refs. 2 and 3 and Ref. 15). The cyclin D-dependent kinases CDK4 and CDK6 phosphorylate RB partially and the process is completed by cyclin E-CDK2 (16, 17). The action of cyclin E-CDK2 appears to depend on prior phosphorylation by the cyclin D-dependent kinases (17). At least 10 serine and threonine residues can become phosp...

p107 and p130 are two closely related proteins that display 30 to 35% identity with the RB polypeptide and share some of its functions. We show that p107 and p130 can both repress Pol III transcription in transient transfection assays or when added to cell extracts. Pull-down assays and immunoprecipitations using recombinant components demonstrate that a subunit of TFIIIB interacts physically with p107 and p130. In addition, endogenous TFIIIB is shown by cofractionation and coimmunoprecipitation to associate stably with both p107 and p130. Disruption of this interaction in vivo by using the E7 oncoprotein of human papillomavirus results in a marked increase in Pol III transcription. Pol III activity is also deregulated in fibroblasts derived from p107 p130 double knockout mice. We conclude that TFIIIB is targeted for repression not only by RB but also by its relatives p107 and p130. The retinoblastoma protein RB has two close relatives, called p107 and p130, to which it is 30 to 35% identical (re-viewed in references 13 and 27). These three are often referred to as the pocket proteins, because most of their homology lies within a bipartite region called the pocket domain. They can each inhibit cell growth and proliferation when overexpressed in tumor cells, an effect that is associated with G 1-specific cell cycle arrest (7, 30, 44, 45). A number of common target proteins have been found to interact with the pocket domains of RB, p107 and p130, including members of the E2F family of cellular transcription factors and the oncoprotein products of several DNA tumor viruses (reviewed in references 10, 15, 27, and 36). As a consequence, there is significant redundancy between the various pocket proteins. This is particularly marked for p107 and p130, which are much more closely related to each other (50% amino acid identity) than they are to RB (30 to 35% identity). A clear demonstration of the redundancy between p107 and p130 was provided by the phenotypes of knockout mice. Thus, animals lacking either p107 or p130 develop normally, whereas animals lacking both of these pocket proteins die within hours of being born (8). In contrast, Rb / mice die at midgestation, displaying defects in both proliferation and differentiation of certain cell lineages (6, 17, 23). This suggests that at least some functions of RB cannot be performed by p107 or p130. This contention is supported by the fact that many tumors contain mutations in Rb, whereas the genes encoding p107 and p130 are not targeted for inacti-vation in cancers. One of the functions of RB that has been demonstrated relatively recently is the regulation of RNA polymerase (Pol) III transcription (reviewed in references 21 and 39). The synthesis of tRNA and 5S rRNA by Pol III in vivo was found to be fivefold more active in primary fibroblasts from Rb / mice than in the corresponding cells from Rb / mice (43). Furthermore , recombinant RB will inhibit the transcription of a range of Pol III templates both in vitro and in transfected cells (5, 22, 43). These effects can...