mTOR-Dependent Regulation of Ribosomal Gene Transcription Requires S6K1 and Is Mediated by Phosphorylation of the Carboxy-Terminal Activation Domain of the Nucleolar Transcription Factor UBF†

Abstract

Mammalian target of rapamycin (mTOR) is a key regulator of cell growth acting via two independent targets, ribosomal protein S6 kinase 1 (S6K1) and 4EBP1. While each is known to regulate translational efficiency, the mechanism by which they control cell growth remains unclear. In addition to increased initiation of translation, the accelerated synthesis and accumulation of ribosomes are fundamental for efficient cell growth and proliferation. Using the mTOR inhibitor rapamycin, we show that mTOR is required for the rapid and sustained serum-induced activation of 45S ribosomal gene transcription (rDNA transcription), a major rate-limiting step in ribosome biogenesis and cellular growth. Expression of a constitutively active, rapamycininsensitive mutant of S6K1 stimulated rDNA transcription in the absence of serum and rescued rapamycin repression of rDNA transcription. Moreover, overexpression of a dominant-negative S6K1 mutant repressed transcription in exponentially growing NIH 3T3 cells. Rapamycin treatment led to a rapid dephosphorylation of the carboxy-terminal activation domain of the rDNA transcription factor, UBF, which significantly reduced its ability to associate with the basal rDNA transcription factor SL-1. Rapamycin-mediated repression of rDNA transcription was rescued by purified recombinant phosphorylated UBF and endogenous UBF from exponentially growing NIH 3T3 cells but not by hypophosphorylated UBF from cells treated with rapamycin or dephosphorylated recombinant UBF. Thus, mTOR plays a critical role in the regulation of ribosome biogenesis via a mechanism that requires S6K1 activation and phosphorylation of UBF.Cell growth (increased cell mass and size) is a prerequisite for proliferation (increased cell number), since a cell will divide only after it has reached a critical mass (38,49,55,58). Thus, factors that govern cell cycle progression must also regulate growth in an interrelated fashion. Cell growth is not however, unconditionally dependent on cell cycle progression, as mutations in the budding yeast Saccharomyces cerevisiae and the fruit fly Drosophila melanogaster that block or disrupt cell division do not necessarily arrest cell growth (34,44). Recent studies have demonstrated that cell growth and cell cycle progression in proliferating mammalian cells, like lower organisms, are also separable processes (8, 50, 63). Thus, detailed knowledge of the biochemical and molecular mechanisms governing cell size will be essential to understanding how the cell division cycle is coupled to growth and how this process is uncoupled during differentiation or is perturbed during diseases associated with deregulated growth. Our knowledge of cell cycle regulatory mechanisms has advanced considerably over the past decade. In contrast, information on the mechanisms of regulating cell growth in mammalian cells is limited.Increased protein synthesis is one of the major anabolic events required for the growth response (28). Recent studies suggest that one of the key nodal points upon which signaling pathwa...

Early loss of mammalian target of rapamycin complex 1 (mTORC1) signalling and reduction in cell size during dominant-negative suppression of hepatic nuclear factor 1-α (HNF1A) function in INS-1 insulinoma cells

“…It is interesting to mention in this context that a recent microarray analysis has also shown that DN-HNF1A expression decreases the transcription of several ribosomal proteins [34]. Targets of S6K1 other than ribosomal protein S6 have been identified, such as the transcription factor cAMP responsive element modulator (CREM)-τ, the RNA splicing/export factor cap binding protein 80 (CBP80) and the eukaryotic translation elongation factor 2 (eEF2) [35], which may mediate additional activities of S6K1 that were hitherto partially unknown. Loss of S6k1 in Drosophila is semi-lethal, and a markedly reduced body size was seen in the surviving adults [36].…”

The balance between rRNA and ribosomal protein synthesis up- and downregulates the tumour suppressor p53 in mammalian cells

“…We demonstrated that a downregulation of rRNA synthesis induced by silencing the POLR1A gene coding for the RNA polymerase I catalytic subunit stabilised p53 without giving rise either to structural changes in the nucleolar components or leakage of the nucleolar proteins to the nucleoplasm. As we found that, according to the available evidence (see reviews by Zhang and Lu, 2009;Deisenroth and Zhang, 2010), the inhibition of the synthesis of rRNA was associated with an increased binding of rpL5 and rpL11 to MDM2, we investigated whether p53 stabilisation also occurred in experimental models in which the synthesis of rRNA and ribosomal proteins was reduced at the same time, such as in cells after serum deprivation or exposure to rapamycin (O'Mahony et al, 1992;Mahajan, 1994;Glibetic et al, 1995;Hannan et al, 2003;Caldarola et al, 2004;Gera et al, 2004;Bilanges et al, 2007). The data obtained, showing no p53 stabilisation in either conditions, indicated that p53 was stabilised only when the reduction of rRNA synthesis was not accompanied by a similar variation in the synthesis of ribosomal proteins, that is, when a relative increase in ribosomal protein availability for MDM2 binding was induced.…”

rRNA gene silencing and nucleolar dominance: Insights into a chromosome-scale epigenetic on/off switch

“…Once the on/off decision has been made, eukaryotic cells then appear to regulate the frequency at which RNA polymerase I initiates on each active gene in order to fine-tune the amount of rRNA produced. Studies in yeast and mammals indicate that fine-tuning is accomplished primarily through signal transduction mechanisms that lead to the post-translational modification of essential RNA polymerase I transcription factors [57][58][59][60][61][62].…”