Regulation of protein synthesis is an important aspect of growth control. RNA polymerase (pol) III plays a key role in this process by catalysing production of tRNA and 5 S rRNA. Growth factors trigger a rapid increase in pol III activity and this is essential for cell proliferation. The transcription factor TFIIIB plays a key role in controlling pol III activity and is a target for regulation by a number of mechanisms. This review will focus on how TFIIIB is targeted by these proteins in response to mitogen stimulation.

Introduction

Tight regulation of protein synthesis is necessary for normal cell growth control. An increase in the production of new proteins, for example, is critical for cell division since progression through the cell cycle will not occur until enough protein has been synthesized. pol III (RNA polymerase III) plays a pivotal role in this process as it catalyses production of a variety of short, untranslated RNA molecules, many of which have essential functions in cellular metabolism. These include tRNA and 5 S rRNA, which are required for protein synthesis, 7SL RNA, which is involved in protein translocation, 7SK which regulates a pol II elongation factor and the U6, H1 and MRP RNAs, which are involved in post-transcriptional processing [1]. Consequently, pol III activity is tightly linked to growth conditions such that transcription by pol III decreases when cells are deprived of serum or nutrients, and increases again upon mitogenic stimulation. Also, transcription of pol III target genes is subject to strict cell cycle control in mammals [2]. Synthesis of tRNA and 5 S rRNA increases within minutes of serum addition but then increases sharply several hours later as cells reach the S-phase [24]. Increased transcription by pol III is a prerequisite for increased protein production and cell proliferation.

Two multi-subunit transcription factor complexes, TFIIIB and TFIIIC, control the action of pol III. When activated, TFIIIB serves to recruit pol III to the appropriate templates and position it at the transcription start site. The action of this factor is aided by TFIIIC, which is required for promoter recognition by binding directly to DNA; it then recruits TFIIIB by protein–protein interactions [5]. TFIIIB is an important control point for regulating pol III transcription. For example, during the cell cycle, TFIIIB is largely repressed as cells enter mitosis and in early G1 [2]. This effect is specific, since TFIIIC and other pol III factors do not display the same fluctuation in cycling cells. This review will focus on describing TFIIIB regulation in the control of mitogen-stimulated pol III transcription.

Repression of pol III transcription by retinoblastoma protein

RB (retinoblastoma protein) is an extremely abundant tumour suppressor that regulates a variety of key transcription factors. In normal cells, RB is involved in constraining growth and proliferation and one of the ways this occurs is by targeting and suppressing pol III transcription in growth-arrested cells [6]. Support for this idea has come from both in vitro and in vivo experiments that show that overexpressing RB can repress pol III transcription [6,7]. Endogenous RB has also been found to control pol III activity when present at physiological concentrations within a cell. Primary embryonic fibroblasts, for example derived from RB-deficient mice, display increased levels of tRNA and 5 S rRNA compared with cells from wild-type mice [6]. Subsequent co-fractionation and immunoprecipitation analyses found that RB could specifically interact with and inactivate the pol III factor TFIIIB [7,8]. When bound by RB, TFIIIB binding to both TFIIIC and to pol III is blocked [8].

A combination of in vitro and in vivo experiments has shown that TFIIIB is targeted for repression not only by RB but also by its close relatives, p107 and p130 [9]. Recombinant p107 and p130 both bind to TFIIIB and repress a variety of pol III-transcribed genes in cell extracts and in transfected cells. Furthermore, endogenous p107 and p130 were found to stably associate with endogenous TFIIIB [9]. Disruption of this interaction markedly increases pol III transcription, and primary fibroblasts from p107−/−p130−/− double-knockout mice display elevated levels of pol III transcripts [9]. The ability to bind and repress TFIIIB is, therefore, a feature of all these pocket proteins, consistent with deletion and substitution analyses that show that regulation of pol III transcription is dependent on the pocket domain [6].

The function of these pocket proteins is subject to cell cycle control and their phosphorylation status fluctuates during this time [10]. It is only in the hypophosphorylated form, found in G0 and early G1, that they can bind and repress TFIIIB [3]. It was demonstrated that RB function is inactivated in late G1 by its hyperphosphorylation by cyclin D- and E-dependent kinases [11]. Phosphorylation of RB causes it to dissociate from its target proteins thereby releasing TFIIIB from repression and allowing it to interact with TFIIIC to recruit pol III and trigger the increase in tRNA and 5 S rRNA that occurs at the G1–S phase transition [2,3]. Thus the phosphorylation status of RB largely dictates the activity of TFIIIB during the cell cycle and, therefore, plays a major role in the growth factor sensitivity of pol III transcription. Although a major increase in pol III activity occurs at the G1–S boundary, an immediate increase in pol III transcription occurs after growth factor stimulation of quiescent cells. This suggests that other mechanisms also exist to execute the rapid increase in pol III transcription that precedes hyperphosphorylation of RB.

Activation of TFIIIB

In response to growth factor stimulation of mammalian cells, TFIIIB is phosphorylated and at least two kinases have been identified which bind to this factor and activate pol III transcription. The ERK (extracellular-signal-regulated kinase) mitogen-activated protein kinases are stimulated in response to mitogens through a signalling cascade involving Ras, Raf and MEK. Once activated, ERK translocates to the nucleus to phosphorylate a number of different transcription factors. TFIIIB has been identified as an important target for regulation by ERK, and this suggests that its ability to stimulate biosynthetic capacity and growth involves direct activation of transcription by pol III. ERK was found to phosphorylate and activate TFIIIB specifically, thereby promoting the assembly of the pol III transcription complex [4]. It has previously been found that members of the mitogen-activated protein kinase family interact with their substrates via specific docking sites known as D-domains and FXFP motifs and subsequent phosphorylation of a downstream Ser/Thr-Pro site [12]. Pol III transcription was compromised by substitutions of ERK docking and phosphoacceptor sites within one of the TFIIIB subunits [4]. Mitogenic stimulation of ERK activity therefore increases cell growth by direct transcriptional activation of 5 S rRNA and tRNA. However, since phosphorylation of TFIIIB is only partially reduced by blocking ERK activity, it is probable that this is not the only kinase involved.

Maximal pol III activity also requires the action of the ubiquitous and highly conserved protein kinase, CK2. Many studies have found an increased activity of this kinase to be associated with cell growth and proliferation [13,14]. An important function of CK2 in cells is to induce pol III transcription, since in both yeast and mammals it has been shown to stimulate synthesis of tRNA and 5 S rRNA [15,16]. CK2 stably interacts with TFIIIB and inhibition of the kinase specifically compromises the binding of TFIIIB to TFIIIC, and thereby inhibits pol III transcription. Through its potent effect on pol III activity, CK2 will probably have a major impact on the biosynthetic capacity of cells and contribute to its ability to promote proliferation.

In addition to being activated by CK2 and ERK, TFIIIB is also a direct target for the proto-oncogene c-Myc. This protein encodes a basic helix–loop–helix leucine zipper transcription factor that has a profound role in growth control and cell cycle progression [17]. Once activated, c-Myc drives a rapid increase in translation, and the fact that c-Myc also has a potent stimulatory effect on pol III transcription provides evidence that the protein synthesis apparatus is a target for the action of c-Myc [18]. Chromatin immunoprecipitation analysis showed the presence of c-Myc at pol III-transcribed genes. This recruitment was found to occur by protein–protein interactions of TFIIIB with the N-terminal transactivation domain of c-Myc [18]. The ability to regulate pol III output may, therefore, be integral to the growth control functions of c-Myc.

Summary

The pol III-specific transcription factor TFIIIB provides a major control for determining cellular activity. As described, the function of several important regulatory proteins may be to regulate the function of TFIIIB, which occurs through a series of protein–protein interactions and covalent modifications. A major challenge will be to identify how this complex array of interactions is co-ordinated to regulate TFIIIB and, therefore, pol III transcription. In addition to determining how protein synthesis and growth are regulated in normal cells, this may also provide an insight into how this becomes deregulated in diseases where protein synthesis is defective.

Genes: Regulation, Processing and Interference: A Focus Topic at BioScience2004, held at SECC Glasgow, U.K., 18–22 July 2004. Edited by I. McEwan (Aberdeen, U.K.), B. White (Glasgow, U.K.), S. Graham (Glasgow, U.K.), S. Roberts (Manchester, U.K.), A. Sharrocks (Manchester, U.K.), D. Black (Organon, U.K.), S. Newbury (Oxford, U.K.), J. Sayers (Sheffield, U.K.) and A. Lloyd (University College London, U.K.).

Abbreviations

     
  • ERK

    extracellular-signal-regulated kinase

  •  
  • pol III

    RNA polymerase III

  •  
  • RB

    retinoblastoma protein

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