Regulation, quality control and homeostasis of ribosome production

Ribosome synthesis devours vast amounts of energy and amino acids in a growing cell. It is therefore easy to understand that ribosome biogenesis has to be tightly regulated.

Prokaryotic cells mainly regulate ribosome synthesis at the level of rDNA transcription and translational feedback mechanisms (for recent reviews see (Wagner, 2002; Dennis et al., 2004; Magnusson et al., 2005; Suthers et al., 2007; Kaczanowska et al., 2007)). Eukaryotic cells regulate ribosome biogenesis basically in the same manner, but the way this is achieved is quite different (see further down).

Each prokaryotic cell has to deal with fast changing environmental conditions. Nutrient depletion, in particular amino acid starvation triggers the so-called “stringent response” on ribosome biogenesis. The binding of deacetylated amino acids to the ribosome, which appear due to amino acid deprivation, causes the synthesis of (p)ppGpp (guanosine 3'-diphosphate 5'-(tri)diphosphate). The induction of (p)ppGPP synthesis can be also a result of carbon or energy source deprivation. Finally, (p)ppGpp binds to the RNA polymerase and shuts down transcription, yet the exact mechanism is still under discussion.

Steady-state or in other words, growth rate regulation enables bacterial cells to adapt to the overall growth conditions. In contrast to the stringent response, steady-state regulation is achieved by varying the number of ribosomes. Feedback loops of r-protein mRNA translation and attenuation or intensification of rDNA synthesis facilitate the change in ribosome number.

Free ribosomal proteins were shown to bind their messenger RNA, thus translational repressing their own expression. This excess of free ribosomal proteins is reflecting an imbalance of rRNA to r-proteins, thereby linking translation to ongoing transcription and r-protein assembly. The molecular signals that result in modification of RNA polymerase activity are still unclear, but (p)ppGpp might again be the main effector molecule. Somehow ongoing translation by functional ribosomes results in a decrease of (p)ppGpp levels and thereby in an increase of RNA polymerase activity.

Eukaryotes seem to have a similar pathway to the stringent response in Bacteria. There is no known small effector molecule like (p)ppGpp, but a specialized protein family was identified (GCN, general control non-derepressible proteins). Upon amino acid deprivation, they initiate a response that leads to a general reduction in protein synthesis, but on the other hand to an upregulation of amino acid anabolism (reviewed in (Hinnebusch, 2005)). Regulation of r-protein expression could be mediated by protein kinase A (PKA), rather than the GCN protein family, since only mutations in the PKA and not in Gcn1p or Gcn4p show a deregulation of expression (Moehle and Hinnebusch, 1991; Klein and Struhl, 1994).

The common pathway in eukaryotes of sensing nutrient availability and other environmental conditions, is mediated by the TOR (target of rapamycin) kinase ((Powers and Walter, 1999) reviewed in (Lempiäinen and Shore, 2009)). The current model of TOR dependent ribosome biogenesis regulation is illustrated in Figure 19 A. The TOR kinase is found in two structural

Introduction

and functional diverse complexes termed TORC1 and TORC2 (TOR kinase complex 1 or 2).

The TORC2 main functions are regulation of the cytoskeleton dynamics and the AGC kinase family (protein kinase A, G, C) (reviewed in (Cybulski and Hall, 2009)). The TORC1 is sensitive to stress and lack of nutrients (mimicked by the drug rapamycin). These stimuli block the activation (phosphorylation) of various effector molecules, like the Sch9p kinase or Sfp1p (Figure 19 A). This in turn shuts down the transcription of r-protein or the Ribi (ribosome biogenesis) regulon genes, which encompasses the genes coding for ribosome biogenesis factors and snoRNAs.

Figure 19. Control of ribosome biogenesis in eukaryotes

(A) The TOR pathway in yeast. The TOR complex 1 (TORC1) and the downstream effector proteins are shown. The upper panel illustrates the situation during growth, the lower panel upon growth inhibition. (B) The regulation of transcription and translation of ribosomal components in mammalian cells. (A) and (B) For simplification, not all pathway components and connections are shown. Question marks indicate poorly understood connections. Further details are elucidate in the main text. (modified from Lempiäinen and Shore, 2009)

Coordination of rDNA transcription and r-protein/Ribi gene transcription is maybe achieved through a common factor to all three RNA polymerases – Hmo1p (UBF1 in higher eukaryotes) (Hall et al., 2006; Berger et al., 2007). In addition TOR was found to directly bind to Pol I promoters at the rDNA locus (Li et al., 2006) and probably regulation the core transcription factor Rrn3p (TIF1A in higher eukaryotes) (Claypool et al., 2004; Mayer et al., 2004). Processing of pre-rRNA and assembly of r-proteins might as well be coupled to transcription of r-protein and Ribi genes. In yeast, the essential Pol II transcription factor Ifh1p is found in a second complex with casein kinase 2, Utp22p, and Rrp7p, termed the CURI complex (Rudra et al., 2007). Utp22p and Rrp7p are both components of the 90S pre-ribosome and necessary for early pre-rRNA processing and assembly events (Baudin-Baillieu et al., 1997b; Dragon et al., 2002b).

On the other hand, it was claimed that ribosomal proteins in HeLa cells are produced in excess, compensated by constitutive degradation of non-assembled r-proteins (Lam et al., 2007). Furthermore, in yeast, inhibition of the TOR kinase leads to severely reduced levels of newly synthesized ribosomal subunits without preceding impairment of Pol I transcription (Reiter, Steinbauer, Philippi et al., 2010, unpublished). Thus, the amount of available free r-proteins could control the rate of ribosome production.

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In contrast to yeast cells, mammals seem to prefer post-transcriptional control of ribosome biogenesis (Figure 19 B). An imbalance in ribosomal subunit stoichiometry causes upregulation of transcription of a subset of mRNAs, containing a 5'-TOP (poly-pyrimidine) motif (Fumagalli et al., 2009). Among these is e.g. rpL11, which was shown to inhibit the transcription factor c-myc (Dai et al., 2007, 2010), but also binds to MDM2 and induces p53 dependent cell cycle arrest (Zhang et al., 2003; Fumagalli et al., 2009).

Furthermore, there is rising evidence that in eukaryotes ribosome biogenesis is regulated in multiple additional layers, like e.g. changes in chromatin structure (Murayama et al., 2008) or plasticity of rDNA repeat numbers (Nomura, 1999).

Many quality control pathways assure that the error-prone process of ribosome biogenesis finally results in functional mature ribosomes. During eukaryotic ribosome biogenesis, misfolded or misassembled precursors are detected, polyadenylated by the TRAMP (Trf4p-Air1/2p-Mtr4p polyadenylation) complex and subsequently degraded by the exosome (among others (Dez et al., 2006; Schneider et al., 2007; Wery et al., 2009)). The ultimate way of regulating a cell's ribosome content is the degradation of ribosomes themselves.

Ribosomes that carry a potentially lethal defect in the rRNA, are normally matured, but become rapidly degraded in the cytoplasm by the so-called NRD (non functional rRNA decay) pathways (LaRiviere et al., 2006; Cole et al., 2009). These pathways are thought to recognize stalled ribosomes and independently degrade the defective SSUs and LSUs, respectively (reviewed in (Lafontaine, 2010)). 18S NRD most probably is initiated by endonucleolytic cleavage of 18S rRNA, followed by complete exonucleolytic digest. 25S NRD on the other hand, is carried out by tagging defective LSUs (associated) components with ubiquitin, followed by proteasomal degradation.

Two specialized forms of autophagy, the ribophagy and PMN (piecemal microautophagy of the nucleus) lead to bulk degradation of mature and pre-ribosomes, respectively, upon nutrient depletion. PMN isn't yet well characterized, though it might result in vacuolar degradation of nucleolar pre-ribosomes (Roberts et al., 2003). Ribophagy most probably is promoted via deubiquitylation of unidentified ribosome associated factors, leading to selective uptake of mature subunits into the vacuole. Like in the NRD pathways, ribophagy of the SSU and the LSU seem to be independently regulated (Kraft et al., 2008; Kraft and Peter, 2008).

Introduction