Regulation of RNA Polymerase III Transcription during Cell Cycle Entry

Abstract

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...

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

“…To this end, transcript levels were compared from matched embryonic fibroblasts of wild-type and RB-knockout mice. Although tRNA gene expression is clearly elevated in the Rb À/À cells, as demonstrated previously (White et al, 1996;Scott et al, 2001), no increase was seen in Brf1 mRNA ( Figure 6, lanes 1 and 2). Similarly, triple knockout (TKO) fibroblasts from Rb À/À p107 À/À p130 À/À mice show elevated tRNA, but normal levels of Brf1 mRNA ( Figure 6, lanes 3 and 4).…”

“…To test this, we investigated whether raising the level of Brf1 can stimulate transcription of tRNA and 5S rRNA genes in cervical cells. Previous work showed that pol III transcription increases markedly at the G1/S phase transition due to a marked increase in TFIIIB activity as it dissociates from RB and p130 (White et al, 1995b;Scott et al, 2001). We therefore examined the response to Brf1 in both G1 and S phases.…”

RNA polymerase III transcription and cancer

“…Inactivation of RB will therefore derepress TFIIIB and allow increased rates of pol III transcription. This was demonstrated clearly with fibroblasts from RB-knockout mice, which show substantially elevated synthesis of tRNA and 5S rRNA when compared to matched wild-type cells (White et al, 1996;Scott et al, 2001). RB is expressed ubiquitously in mammals, and it is believed that RB function must be compromised for a cancer to develop (Weinberg, 1995).…”

“…Again, this reflected a specific increase in the levels of mRNAs encoding all five subunits . The mechanisms responsible for raising TFIIIC2 expression have yet to be determined, but they do not seem to reflect a simple response to accelerated proliferation Scott et al, 2001).…”

Pocket proteins and cell cycle control

“…G0 cells can be distinguished from those in G1 by their diminished RNA content, which reflects diminished production of ribosomal RNAs and tRNAs (Lajtha, 1963;Ladd et al, 1997;Ren and Rollins, 2004). Pocket proteins may contribute to the G0 state by repressing ribosomal RNA and tRNA gene expression, whereas pocket protein phosphorylation may restore ribosomal RNA and tRNA levels to effect the G0-G1 transition (Cavanaugh et al, 1995;White, 1997;Hannan et al, 2000;Scott et al, 2001).…”