RNA Polymerase III Transcription Factor IIIB Is a Target for Repression by Pocket Proteins p107 and p130

Abstract

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...

Deregulation of RNA polymerase III transcription in cervical epithelium in response to high-risk human papillomavirus

“…Thus, induction of Brf1 could be responsible for the observed abundance of tRNA and 5S rRNA in cervix infected with HPV16. However, an alternative explanation is provided by the fact that the E6 and E7 products of HPV16 can both derepress TFIIIB, by neutralizing p53 and RB, respectively (Larminie et al, 1999;Sutcliffe et al, 1999;Stein et al, 2002). Viral integration stimulates production of these oncoproteins and also correlates in cultured W12 cells with increased levels of pol III transcripts and Brf1 mRNA.…”

“…Inactivation of either of these tumour suppressors can cause elevated rates of pol III transcription in vivo (Felton-Edkins et al, 2003). For example, neutralizing RB with the E7 oncoprotein of HPV16 can stimulate expression of a pol III reporter in transfected fibroblasts, but this response is abolished by mutations that prevent the RB/E7 interaction (Larminie et al, 1999;Sutcliffe et al, 1999). Similarly, HPV16 E6 oncoprotein can relieve repression of a pol III reporter by p53 transfected into SAOS2 cells (Stein et al, 2002).…”

RNA polymerase III transcription can be derepressed by oncogenes or mutations that compromise p53 function in tumours and Li-Fraumeni syndrome

“…This function is compromised by a number of mutations which arose naturally in human carcinomas Sutcli e et al, 2000;White et al, 1996). Pol III transcription is released from repression by E7, E1A and SV40 large T antigen, three viral oncoproteins which bind to RB (Larminie et al, 1999;Sutcli e et al, 1999;White et al, 1996). In a high proportion of cancers, the function of RB is compromised due to its hyperphosphorylation; this can be caused by overexpression of cyclin D or loss of p16, a speci®c inhibitor of cyclin D-dependent kinases (Bates and Peters, 1995;Hunter and Pines, 1994).…”

RNA polymerase III transcription and cancer

“…Several DNA tumour viruses encode oncoproteins that neutralize RB by binding to its pocket (Vousden, 1995). These include the E1A product of adenovirus, the E7 product of HPV and the large T antigens of SV40 and polyomavirus, all of which have been shown to release TFIIIB from repression and thereby stimulate pol III transcription in vitro and in vivo (White et al, 1996;Larminie et al, 1999;Sutcliffe et al, 1999;Felton-Edkins and White, 2002). Substitutions in E7 that preclude its interaction with RB also block its effect on a pol III reporter (Larminie et al, 1999;Sutcliffe et al, 1999).…”

“…These include the E1A product of adenovirus, the E7 product of HPV and the large T antigens of SV40 and polyomavirus, all of which have been shown to release TFIIIB from repression and thereby stimulate pol III transcription in vitro and in vivo (White et al, 1996;Larminie et al, 1999;Sutcliffe et al, 1999;Felton-Edkins and White, 2002). Substitutions in E7 that preclude its interaction with RB also block its effect on a pol III reporter (Larminie et al, 1999;Sutcliffe et al, 1999). Probably the most prevalent mechanism by which RB function is compromised in tumours is through its deregulated hyperphosphorylation by cyclin D-and E-dependent kinases (Hunter and Pines, 1994;Bates and Peters, 1995;Sherr, 2001).…”