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). Translation initiation typically requires that the initiation complex scans a mRNA until finding an appropriate codon (cf I.A.2.). However, some mRNA displays leaky scanning, where the first AUG codon is not always selected for initiation (Kozak, 2002). Indeed, the sequence context has been shown to be critical for initiation efficiency, directly influencing translation rate and start codon choice. The optimal sequence, also known as the Kozak sequence (GCCA/GCCaugG), has been shown to yield 20 times more protein than weak sequences. Interestingly, the presence of a purine at the [-3] position and a guanine at [+4] contributes the most to initiation efficiency (Kozak, 1986). It was postulated that AUG codon selection was enhanced by ribosome stalling mediated by base specific interactions. Indeed, uS19 interacts with both a U or G at the [+4] position, though other bases were not tested. More interestingly, uS7 interacts with a U at the [-3] position but not with a G, possibly underlying base discrimination mechanisms for AUG selection. Similarly, eS26 was shown to bind a U between positions [-8] and [-11] (Pisarev et al., 2006). Strikingly, ribosomes lacking eS26 were shown to translate mRNA with weak Kozak sequences more than complete ribosomes. Those mRNA are enriched for specific stress response pathways. Cells lacking eS26 expression constitutively activate those response pathways suggesting that incorporation of eS26 in the ribosome may be a mechanism that modulates the translational landscape (Ferretti, 2015, 10th EMBO conference on ribosome synthesis, unpublished data).
Trans-acting factors control translation
Ribosomal proteins are abundant, bind nucleic acids unspecifically, are susceptible to aggregation, and populate all cellular components. Those properties hinder the analysis of their interactions with other molecules. Indeed, they have historically been discarded as contaminants from classical molecular biology experiments, up to even recent mass-spectrometry analysis workflows (Dahlberg et al., 2003; Mellacheruvu et al., 2013). Indeed, while many translation regulators are known, their regulatory mechanism is only poorly understood.
RNA binding factors
Experimental studies have identified more than 800 mRNA binding proteins in mammals, many of which potentially regulate translation (Baltz et al., 2012; Castello et al., 2012). Several such regulators have already been characterized which display context dependant activity. The Drosophila sex lethal protein represses translation in females (cf III.A.1), and the Iron Response Element binding protein (IRE-BP) does the same in the absence of iron (Hentze and Kühn, 1996; Moschall et al., 2017). Similarly, the expression of 15-lipoxygenase is repressed by hnRNPK and hnRNPE1 at the translational level in erythroid precursors (Ostareck et al., 2001).
Extra-ribosomal functions of r-proteins
Once considered full-time constituents of the ribosome, r-proteins are now studied for their participation in many cellular processes. While a substantial part of their contribution to cell metabolism has been attributed to their ability to alter ribosome behaviour with consequences on protein synthesis, it has been known for a long time that some ribosome-free r-proteins also carry regulatory activities, consequently termed “extra-ribosomal functions”. The increasing number of such examples over the last decades led to the idea that those would be a general feature of r-proteins. However, the existence of a genuine extra-ribosomal function is hard to prove and harder to study. Three criteria must be met (Warner and McIntosh, 2009). 1) The r-protein interacts with a non-ribosomal component. 2) This interaction must have a physiological effect. 3) It occurs away from the ribosome. Since tampering with an r-protein is expected to cause translation defects, with extensive effects on physiology, careful attention must be paid to specifically attribute observations to the extra-ribosomal function alone. This can be difficult as most of these functions are involved in the regulation of ribosome biogenesis, or response to nucleolar stress, with expected effects on ribosomal activity. Solving this conundrum is no small feat, explaining why despite numerous observations implicating r-proteins in various processes, readily characterized extra-ribosomal functions are still scarce.
When are r-proteins free?
The idea that r-proteins carry regulatory functions out of the ribosome is problematic on several levels. Indeed, extensive studies about their role in ribosome biogenesis yielded the notion that their existence is very tightly controlled, and that they are very unstable on their own. Thus, it seems important to question under which circumstances can r-proteins exert functions out of the ribosome. In the next section, several studies are mentioned which use the r-protein mediated stabilisation of p53 as a readout for the ability for r-proteins to perform their extra-ribosomal functions. For clarity purpose, the underlying mechanism of this stabilisation will be detailed in a further section (cf. III.B.2).
r-proteins participate in cell metabolism
Numerous extra-ribosomal functions have been discovered over the last years, and the number is still on the rise. Several reviews have made attempts to provide a list of these (Bhavsar et al., 2010; Wool, 1996; Zhou et al., 2015). It should however be noted that authentic extra-ribosomal functions are often intermingled with potential functions suggested by circumstantial lines of evidence (for instance uL16 was attributed the extra-ribosomal function “Autism” while eL13 was assigned an extra-ribosomal function for being up-regulated in response to DNA damage). Those should only be considered with caution, as the demonstration of an extra-ribosomal function requires stringent criteria (Warner and McIntosh, 2009). Nevertheless, several of them have been readily characterized, implicating extra-ribosomal functions in a number of metabolic events. Many such functions are the prerogative a single r-protein, and the range of affected processes is broad. For instance an extra-ribosomal function was attributed to eS19 as a monocyte chemotactic factor (Yamamoto, 2000). For these reasons, the next chapter will not contain an exhaustive list of extra-ribosomal functions, but rather describe those that are general features of r-proteins.
Table of contents :
Table of contents
Table of figures
Table of abreviations
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.
C. Materials and methods
II. Editing essential genes: a marker independent CRISPR mutagenesis strategy.
C. Complementary data
III. Mutation of a single amino-acid on ribosomal protein uL11 generates a Minute-like phenotype in Drosophila.
uL11K3A assembles into functional ribosomes
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?