Mutation of a single amino-acid on ribosomal protein uL11 generates a Minute-like phenotype in Drosophila

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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.

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.

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.

The Drosophila Minute mutants

Ribosomal protein gene loss of function alleles have been studied for almost a hundred years in Drosophila, where they are known as “Minute” mutations. They have puzzled researchers for decades. Indeed, mutations at more than 50 loci in the genome gave rise to similar phenotypes sharing the same genetic properties (Schultz, 1929). The latter were extensively studied, providing exceptional insight about the requirements for ribosomal protein genes in a developing organism. All Minute mutations are dominant and lethal when homozygous. Minute mutants were first described for displaying thin and short (minute) bristles, together with prolonged development (Brehme, 1939). The vast majority of Minute mutations strongly impact viability and fertility, to the point that several Minute loci could only be identified through transient aneuploidy experiments and were proposed to be “extreme Minute” (Lindsley et al., 1972). Interestingly, the combination of different Minute mutations shows no cumulative effect on bristle length or development length. Additionally, Minute mutations display the same interactions with genetic modifiers. These data led to the hypothesis that Minute mutations affected different components of a single “Minute reaction”, which requires all Minute genes to function. Furthermore, in triploid flies, these mutations were found to be recessive to two copies of a wild type allele. On the opposite, two copies of a Minute allele were lethal in presence of a single wild type allele. These experiments showed that the dominance of Minute mutations is the consequence of haplo-insufficiency. Thus, the core Minute phenotypes were attributed to the limitation of the “Minute reaction”, consequence of the reduction in expression of one of its components (Schultz, 1929). Minute loci have been characterized over time and the Minute genes have now been identified to encode r-proteins with very few exceptions (Cook et al., 2012; Marygold et al., 2007). Indeed, all the characteristics of Minute mutations are consistent with the “Minute reaction” corresponding to the synthesis of mature ribosomes.

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).

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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.

Table of contents :

Table of contents
Table of figures
Introduction
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?
3
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
Results
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?
Bibliography 

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