Mechanistic analysis of RNA polymerase III regulation by the retinoblastoma protein

Abstract

(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...

RNA polymerase III transcription and cancer

“…A far more universal route to deregulate pol III transcription was suggested by the discovery that this system can be strongly repressed by the retinoblastoma tumour suppressor RB (White et al, 1996). Several groups have shown independently that overexpressing RB can potently inhibit pol III transcription in transfected cells or in vitro (White et al, 1996;Chu et al, 1997;Larminie et al, 1997;Hirsch et al, 2000). This is due to the binding of RB to TFIIIB, which blocks its interactions with TFIIIC2 and pol III (Larminie et al, 1997;Sutcliffe et al, 2000).…”

“…Several groups have shown independently that overexpressing RB can potently inhibit pol III transcription in transfected cells or in vitro (White et al, 1996;Chu et al, 1997;Larminie et al, 1997;Hirsch et al, 2000). This is due to the binding of RB to TFIIIB, which blocks its interactions with TFIIIC2 and pol III (Larminie et al, 1997;Sutcliffe et al, 2000). Inactivation of RB will therefore derepress TFIIIB and allow increased rates of pol III transcription.…”

“…In all, 98% of such mutations involve the large pocket domain of RB, which spans residues 393-928 (Harbour, 1998). This region is necessary and sufficient for RB to bind TFIIIB and repress pol III transcription (White et al, 1996;Chu et al, 1997;Larminie et al, 1997). Accordingly, this function is ablated by substitutions or small deletions in the pocket that arose naturally in tumours (White et al, 1996;Brown et al, 2000;Felton-Edkins et al, 2003b).…”

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

“…Again, Brf1 mRNA levels remained unchanged, although tRNA expression is elevated in the knockouts (Figure 6, lanes 5 and 6), as shown previously by nuclear run-on analysis (Cairns and White, 1998). Synthesis of tRNA increases in both sets of knockouts because RB and p53 bind to TFIIIB to repress pol III transcription directly (Cairns and White, 1998;Larminie et al, 1997). However, Brf1 expression seems not to change under these conditions.…”

Mutations in the β-propeller domain of the Drosophila brain tumor (brat) protein induce neoplasm in the larval brain

“…The calibration bars in (a) and (j) indicate 100 mm, and (a ± g) and (j ± r) are of the same magni®cation. In (h) and (s) the bars indicate 50 mm, and (h,i,s) and (t) are of the same magni®cation loss of Rb and p53 function causes substantial increase in RNA polymerase I and III transcriptional activity (Nasmyth, 1996;White et al, 1996;Larminie et al, 1997;Cairns and White, 1998). It is possible that one way in which tumor cells are able to achieve an increase in cell growth is by increasing their ability to synthesize proteins through mutation of genes such as Rb and p53 that normally function to repress RNA polymerase I and III transcription (for review see Nasmyth, 1996;Larminie et al, 1998;White, 1997).…”