Survival of a cell critically relies on its ability to respond to environmental signals. Consequently, living organisms sense and react to the availability of nutrients. In eukaryotes, nutrient-dependent cell growth is governed by the target of rapamycin (TOR) signal transduction pathway which is highly conserved from yeast to humans (reviewed in reference 86
). Rapamycin (RAP) is a macrocyclic lactone, which inhibits the activity of TOR-kinase-containing protein complex 1 (TORC1). TOR inactivation impairs various anabolic processes, including general translation, transcription of many genes, and ribosome biogenesis. It triggers catabolic processes like protein and RNA degradation, as well as autophagy. Clearly, TOR signaling determines the fate of the eukaryotic cell, and it is important to understand the molecular details of this central control mechanism. For consistency, we will focus in this introduction on findings in the model eukaryote Saccharomyces cerevisiae
(here, called yeast), unless stated otherwise.
One important target of TOR control is ribosome biogenesis (reviewed in reference 50
). Ribosome production relies on the concerted action of all three DNA-dependent RNA polymerases (Pols I, II, and III) to produce equimolar amounts of the structural components of the ribosome, the four ribosomal RNAs (rRNAs) and the more than 70 ribosomal proteins (r-proteins). Furthermore, more than 150 auxiliary factors participate in ribosome maturation (reviewed in references 18
, and 72
). TOR signaling has been shown to affect this complex process on several levels: (i) transcriptional regulation of all three polymerases, (ii) translation initiation, (iii) RNA processing, and (iv) internuclear and nucleo-cytoplasmic transport processes. These effects are described below.
(i) Early analyses have demonstrated that amino acid depletion and impaired TOR signaling downregulate RNA Pol II-dependent transcription of r-protein genes (58
). Genome-wide transcription profiling using microarrays and bioinformatics confirmed that r-protein genes define a coregulated cluster, termed the RP regulon (8
). These studies also demonstrated that the transcriptional response of the RP regulon to rapamycin treatment and various other environmental changes was similar to that of a large group of other genes involved in ribosome biogenesis, termed the Ribi regulon. Subsequent work identified a subset of transcription factors specifically binding to DNA elements in the promoter regions of RP- and Ribi-regulon-controlled genes in a TOR-dependent manner (35
). Interestingly, the Ribi regulon included genes belonging to the RNA Pol I and RNA Pol III transcription machineries (21
), thus providing a link between the different nuclear RNA polymerase systems.
The results of in vivo
]methionine pulse-labeling experiments suggested that RNA Pol I transcription and rRNA processing are strongly impaired shortly after TOR inactivation (58
). Since then, various studies have explored the molecular mechanism underlying this observation. It could be shown that the amount of initiation-competent complex of RNA Pol I with the essential transcription factor Rrn3 is reduced in rapamycin-treated cells (11
). This observation correlated well with a reduced RNA Pol I association with the rRNA gene locus under these conditions (11
). Further evidence has been provided that the RNA Pol I-Rrn3 complex is a primary target of TOR signaling. Thus, it has been shown that the expression of a nondissociable fusion protein composed of the RNA Pol I subunit Rpa43 and Rrn3 attenuates the transcriptional inactivation of the three different RNA Pols upon TOR inactivation (41
). In mammals, TOR-dependent changes in the phosphorylation pattern of the Rrn3 homologue transcription initiation factor 1A (TIF-IA) correlate with impaired rRNA gene transcription (51
Recently, the HMG-box protein Hmo1 has been shown to bind to the RNA Pol I-transcribed region of the ribosomal DNA (rDNA), as well as to a subset of the r-protein gene promoter regions (4
). These observations made Hmo1 a potential candidate mediating the cross talk between RNA Pol I and RNA Pol II. Additionally, it has been reported that rapamycin treatment leads to Hmo1 dissociation from its genomic target sites (4
Another factor which may be directly involved in downregulation of RNA Pol I and RNA Pol III transcription following rapamycin treatment is TORC1 itself. A nuclear fraction of TORC1 associates with the rRNA gene promoter and the 5S rRNA gene locus under normal growth conditions but leaves the nucleus in the presence of rapamycin or upon nutrient deprivation (42
). Nuclear localization of TORC1 seems to be important for phosphorylation and, thus, inactivation of the RNA Pol III transcriptional repressor Maf1 (84
). A target for TORC1-mediated phosphorylation within the RNA Pol I transcription machinery, however, has yet to be identified. Noticeably, nuclear-cytoplasmic shuttling has also been observed for mammalian TOR (38
(ii) Rapamycin treatment for only 15 min reduces cellular protein production to less than 50% (2
). One downstream consequence of rapamycin treatment is the activation of Gcn2 kinase which, in turn, phosphorylates the α subunit of eukaryotic initiation factor 2 (eIF2α), thus inhibiting translation initiation (9
). TOR inactivation further affects translation initiation by the degradation of elF4G, an essential protein required for mRNA translation via the 5′ cap-dependent pathway in yeast (5
). However, the exact degradation pathway remains unknown. Interestingly, in mammals TOR controls translation initiation at a similar step by phosphorylation of elF4E-binding protein under normal growth conditions. Upon rapamycin treatment elF4E-binding proteins are dephosphorylated and bind to elF4E, which in turn can no longer act in translation initiation (3
(iii) As mentioned above, rRNA processing is severely impaired in cells treated with rapamycin (58
). This could be due to direct TOR-dependent inactivation of ribosome biogenesis factors and/or to the transcriptional downregulation of genes controlled by the Ribi regulon, leading to rapid depletion of protein factors involved in rRNA maturation. Examples that endogenous protein levels of ribosome biogenesis factors are quickly reduced in the course of rapamycin treatment are Nog1 and Nop7, the amounts of which drop in good correlation with the decrease in the respective mRNA level (32
). Nevertheless, rRNA maturation defects are observed significantly before these factors become limiting.
Impaired rRNA processing is not the only example for TOR-mediated effects on RNA metabolism. TOR inactivation also destabilizes a subset of mRNAs, due to either reduced 3′ polyadenylation or accelerated deadenylation and to 5′ decapping (1
). It is not understood, however, why only certain mRNAs are targeted and by which of the different mechanisms they are eventually destabilized.
(iv) Finally, recent work has provided evidence that TOR signaling is involved in the control of preribosomal transport processes (32
). Nucleolar entrapment of ribosome biogenesis factors early after rapamycin treatment has been observed and has been correlated with the cessation of late rRNA maturation steps or the inhibition of nucleo-cytoplasmic translocation of preribosomes.
Taken together, these observations indicate that TOR inhibition appears to affect ribosome biogenesis on many different levels. However, it has become increasingly difficult to distinguish between primary and secondary effects since the multiple processes leading to mature ribosomes appear to be intimately linked. Here, we performed an in-depth analysis of yeast cellular phenotypes after 15 min of rapamycin treatment. We found that the strong decrease in rRNA production is not closely coupled to the shutdown of RNA Pol I transcription. A significant amount of RNA Pol I remains associated with the rRNA gene upon TOR inactivation, is mobile, and produces transcripts. Furthermore, rDNA interaction with the RNA Pol I transcription factor Hmo1 and other components of the RNA Pol I transcription machinery is unaltered after short times of rapamycin action. Instead, we observe that r-protein production is severely and specifically impaired under this condition. Additionally, short incubation of yeast cells in the presence of cycloheximide (CHX), an inhibitor of eukaryotic protein synthesis, leads to rRNA production defects similar to the rapamycin-mediated maturation phenotype. Both cycloheximide treatment and, in particular, depletion of single r-proteins also provoke nucleolar entrapment of ribosome biogenesis factors. Thus, we conclude that rapid depletion of the endogenous pool of free r-proteins is sufficient to explain various prominent phenotypes observed in the quick response to TOR inactivation.