Ribosome biogenesis as a conductor of cell metabolism

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Ribosome biogenesis is monitored at several stages of cell life

As previously demonstrated, ribosome biogenesis activity is tightly regulated and can be inhibited a number of ways in times of metabolic distress. Thus, ribosome synthesis can be considered a proxy for cell health. Indeed, several mechanisms monitor the state of ribosome biogenesis and regulate cell fate in accordance.

Cell cycle commitment

One such example is the progression into the cell cycle. Indeed, the transition from G1 phase to S phase, implies a commitment to cell division, and is subject to extensive regulation. Passing the G1/S checkpoint requires to reach a critical size, and the same condition applies for the G2/M checkpoint (for review: Barnum and O’Connell, 2014). As ribosomes constitute the largest part of the cell material, their abundance may serve to evaluate cell size. In turn, ribosome number may be measured through translation capacity. Indeed, it has been shown that in yeast, the depletion of many translation factors and tRNA biosynthesis genes induce an arrest of the cell cycle in G1 phase, suggesting that G1 to S phase transition is translation-dependent (Yu et al., 2006). Indeed, translation of the yeast Cln3p was shown to be required to pass this checkpoint (Barbet et al., 1996). Cln3p is an extremely unstable protein, so its accumulation requires intense translation (Tyers et al., 1992). In addition, the presence of an upstream open reading frame (uORF) in the 5’ UTR of the Cln3p mRNA represses its translation when the amount of ribosomes is limited (Polymenis and Schmidt, 1997).
Thus, accumulation of Cln3p is only possible after ribosome number reaches a certain point. Interestingly, a study pointed out that depletion of an rRNA processing factor triggered defects in cell cycle progression before the number of ribosomes or translation capacity started to dwindle, in a Cln3p-independent manner. These data suggest that ribosome biogenesis activity may also be monitored at the level of newly synthetized subunits to trigger cell cycle progression (Bernstein et al., 2007). In addition, the depletion of r-proteins in yeast caused stage specific cell-cycle arrest. Many of them caused G1 phase arrest, consistent with monitoring of either ribosome biogenesis or steady-state levels. Interestingly, nine r-proteins of the large subunit triggered an arrest in G2 phase, suggesting that they are required at the G2/M checkpoint. This specific defect could result from another mechanism than G1 arrest. Strikingly, all nine r-proteins cluster on the solvent side of the 60S subunit, where they could interact with non-ribosomal factors. Thus, they may be needed either as part of “specialized” ribosomes, or they could participate in cytoplasmic export of G2 phase-specific factors, through “ribosome riding” (cf. II.B.2) (Thapa et al., 2013).

Ribosome biogenesis drives cellular organization

The nucleolus is a membrane-less organelle, thought to form upon coalescence of ribosome biogenesis factors around the site of rDNA transcription. It has thus been described as an organelle made by the act of building ribosomes (Mélèse and Xue, 1995). One of its functions is to concentrate ribosome biogenesis factors at the site of ribosome production, but the nucleolus also plays an important role in nuclear organization. Not only does it separate many proteins from the nucleoplasm, it also recruits specific chromatin domains shaping genomic spatial organization and therefore gene expression. Importantly, the nucleolus is a highly dynamic structure, which disappears and reassembles during each cell cycle (cf. II.A.2). Being the site of ribosome synthesis, it is also very responsive to stress. Indeed, many cues that inhibit ribosome biogenesis trigger morphological changes, up to sheer disruption of the nucleolus, and these phenotypes are termed “nucleolar stress” (Boulon et al., 2010). Through this mechanism, ribosome biogenesis impairments impact the whole nuclear organization, causing massive changes in both gene expression and cell metabolism.

The nucleolar associated domains

The nucleolus forms around active rRNA genes, which can be found in the fibrillar centre in a de-condensed state (cf I.B.I). Meanwhile, inactive rDNA genes can be found in a layer of heterochromatin that surrounds the nucleolus, and that localization is required for rDNA stability (Guetg et al., 2010). But rDNA repeats are not the only residents of this peri-nucleolar heterochromatin layer. Indeed, many chromosomal loci are recruited in there, following a reproducible pattern. For instance, the Y and inactive X chromosomes associates preferentially to the peri-nucleolar layer in humans (Bobrow et al., 1971; Zhang et al., 2007). These Nucleolus Associated Domains (NADs) contain a high amount of satellite DNA, notably centromeric, and pericentromeric sequences (Németh and Längst, 2011). In Drosophila , their recruitment is mediated by the nucleolar protein Modulo, and is necessary to maintain the silencing of repetitive regions and maintain proper chromosome segregation during mitosis (Padeken et al., 2013). Telomeric sequences have also been shown to associate with nucleolar heterochromatin. In Arabidopsis, this association requires uc1, and its disruption results in drastic telomere shortening. Nucleolar heterochromatin also contains RNAPolII genes and transposable elements, which are maintained in a transcriptionally repressed state (Pontvianne et al., 2016).
NADs also contain a high amount of RNAPolIII genes, among which 5S rRNA, tRNA and U6 snoRNA genes (Németh and Längst, 2011; Thompson et al., 2003). Those have been shown to induce silencing of nearby RNAPolII promoters. This repression is dependent on active transcription by RNAPolIII and indeed, it is alleviated upon inhibition of ribosome biogenesis. As the loss of nucleolar localization correlated with the de-repression of proximal RNAPolII genes, it was postulated that tRNA silence nearby genes by changing their sub-nuclear localization (Wang et al., 2005). Interestingly, tRNA genes have also been shown to display insulator properties, and prevent the spread of heterochromatin. Unlike their function in proximal gene silencing, this property is independent of transcription by RNAPolIII (Raab et al., 2012). Indeed, the minimal requirement for this heterochromatin barrier activity is the binding of the pre-assembly complex component TFIIIC, which binds the B box on the tRNA genes internal promoter (Simms et al., 2008). As many tRNA genes cluster near the pericentromeric heterochromatin, their insulator property is thought to be necessary to delimit its boundaries (Noma et al., 2006). Another property of tRNA genes is their ability to establish cohesion points with the sister chromatin, further participating to genome stability. Importantly, this cohesion requires not only the binding of TFIIIC, but also active transcription by RNAPolIII (Dubey and Gartenberg, 2007).

Phenotypic consequences of ribosome biogenesis defects

Considering the absolute necessity for protein synthesis at the cellular level, it comes as no surprise that mutations affecting ribosome biogenesis are associated with dire phenotypes. Most of them adversely impact viability and fertility, but they also generate a very broad range of phenotypes, some of which can be hard to link with translation. While there is a considerable amount of data about the specific effects of ribosome impairments, some general features do emerge.

r-protein mutations in S. cerevisiae

The study of S. cerevisiae phenotypes upon ribosome biogenesis impairments provides insight about its importance at the cellular level. It comes as no surprise that the complete loss of most ribosomal proteins prevents proliferation. Indeed, 64 out of 79 r-protein genes are essential, and their conditional inhibition triggers the arrest of the cell cycle at specific checkpoint. They can however be studied by using heterozygous deletion strains. In most cases, they display a slow growth phenotype (Steffen et al., 2012; Thapa et al., 2013). Molecular study of the effect of their depletion on ribosome synthesis showed that the lack of any essential r-protein impairs the production of mature ribosomes. Indeed, mutant cells accumulate pre-subunits stalled at different maturation stages depending on the lacking r-protein (Ferreira-Cerca et al., 2005; Pöll et al., 2009). In contrast, non-essential r-proteins are not required for the production of mature ribosomes. They do however take part in its translational function, and their mutations most often result in very slow growth phenotypes (Baronas-Lowell and Warner, 1990; Briones et al., 1998; DeLabre et al., 2002; Peisker et al., 2008; Remacha et al., 1995). Thus, the reduction in growth rate is thought to result from a limitation of translation capacity.

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Ribosomes regulate gene expression: the second lives of the housekeepers

The idea that some genes are dedicated to the cell’s basic metabolism has been longstanding. These housekeeping genes would be constitutively expressed in all cells, at all times (Eisenberg and Levanon, 2013). Logically, they would be essential for cell survival and require no regulation mechanism whatsoever. Ribosomal biogenesis components perfectly fitted that idea, and were thus considered housekeepers. Unfortunately, “nature was not designed to make life easy for biologists” (Tudge, 2006), which came to realize that even the housekeepers have days off. Indeed, beside doing the cell’s laundry, housekeeping genes have a life of their own, and engage in many activities. Thus began the quest for biologists to discover what the housekeepers do when they are not polishing the silverware (after re-counting the silver spoons, though).

Ribosome-mediated regulation of translation

In the absence of powerful techniques to study proteins, mRNA levels have long been used as proxies to estimate protein abundance. The advent of quantitative mass spectrometry and its tremendous improvements in the recent years opened new perspectives in gene expression research (Ong and Mann, 2005). Notably, the correlation between mRNA and corresponding protein levels were soon found to be surprisingly low. Indeed, depending on organisms and environmental context, mRNA abundance only explain around 40% of the variation in protein levels, suggesting that translation and degradation are major components of gene expression regulation (Abreu et al., 2009; Vogel and Marcotte, 2012). Apprehending the importance of translation regulation has raised concerns, as it seems to render transcription regulation somewhat superfluous. However, a study showed that upon drug treatment, differentially expressed mRNA correlated with protein levels more than steady mRNA (Koussounadis et al., 2015). These data imply that, as expected, transcriptional regulations are reflected at the protein level. Indeed, it has been proposed recently that under steady-state conditions, protein levels could be estimated from mRNA levels by applying to each gene an RNA-To-Protein (RTP) ratio. Strikingly, this ratio was found to vary by orders of magnitude between genes, but to be independent of cell type (Edfors et al., 2016; Silva and Vogel, 2016). Paradoxically, these theories question in turn the necessity for translational regulation. It should however be considered that they spawn from high-throughput analyses and reflect general trends. As such, they do not account for individual genes or gene groups. Furthermore, it is questionable whether they still hold true under stress induction, when ribosome biogenesis is heavily altered. Indeed, an exciting hypothesis would be that regulation of transcription and translation serve different purposes. For instance, it is believed that translational regulation has a much quicker impact on metabolism that transcriptional regulation, and is thus more fit to accommodate stress response mechanisms.

Ribosomes interact with mRNA cis-regulatory elements

While many mechanisms tune general translation through the number of ribosomes (cf. II.A), it is important to note that translational activity can be regulated in a mRNA-specific manner. The affinity of the ribosome for specific mRNA, and the efficiency of translation are critical parameters in gene expression. Indeed, cells contain a finite pool of ribosomes and translation factors, for which mRNAs must compete (Chu et al., 2011; Raveh et al., 2016). Accordingly, their untranslated sequences abound with cis-regulatory elements which influence ribosome recruitment or initiation rate, and allow spatiotemporal control of protein synthesis (For review: Araujo et al., 2012).

Table of contents :

I. The ribosome
A. Ribosome and translation
1. Composition of the ribosome
2. Translation initiation
3. Translation elongation
4. Translation termination
B. Ribosome biogenesis
1. Ribosomal RNA transcription
2. Ribosomal RNA processing
3. rRNA modifications and snoRNPs
4. Ribosomal proteins biogenesis
5. Ribosomal protein assembly
6. Pre-ribosomal factors and quality control
7. tRNA biogenesis
II. Ribosome biogenesis as a conductor of cell metabolism
A. Metabolic cues dictate the activity of ribosome synthesis
1. Cell growth and proliferation signals
2. The cell cycle
3. Energy levels
4. Metabolic stress
B. Ribosome biogenesis regulates homeostasis
1. Ribosome biogenesis is monitored at several stages of cell life
2. Ribosome biogenesis drives cellular organization
3. Phenotypic consequences of ribosome biogenesis defects
III. Ribosomes regulate gene expression: the second lives of the housekeepers
A. Ribosome-mediated regulation of translation
1. Ribosomes interact with mRNA cis-regulatory elements
2. Trans-acting factors control translation
3. Ribosome heterogeneity
B. Extra-ribosomal functions of r-proteins
1. When are r-proteins free?
2. r-proteins participate in cell metabolism
3. r-proteins are bound to chromatin
IV. Regulation of ribosome homeostasis by uL11 and Corto
A. The ribosomal protein uL11
1. General features of Drosophila uL11
2. Known functions of uL11 on and off the ribosome
3. Methylation of uL11
B. The transcription factor corto
1. General features of Corto
2. corto participates in epigenetic maintenance of segmental identity during development.
3. corto may be involved in developmental homeostasis
V. Presentation of the thesis project
I. Ribosomal protein uL11 tri-methylated on lysine 3 binds broad genomic regions and displays an exclusion pattern with Corto on chromatin.
A. Results
B. Discussion
C. Materials and methods
II. Editing essential genes: a marker independent CRISPR mutagenesis strategy.
A. Overview
B. Article
C. Complementary data
III. Mutation of a single amino-acid on ribosomal protein uL11 generates a Minute-like phenotype in Drosophila.
A. Results
uL11K3A assembles into functional ribosomes
B. Discussion
C. Materials and methods
Discussion and perspectives
What is the function of uL11K3me3 and CortoCD on chromatin?
Is uL11K3me3 involved in epigenetic regulation of gene expression?
The N-terminal domain of uL11 carries a critical function in Drosophila
The methylation of uL11 lysine 3 is not essential in Drosophila.
Is the function of uL11 lysine 3 methylation strictly transcriptional?
uL11 as an amplifier of ribosome biogenesis?


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