Contrôle épigénétique de l'homéostasie de la biogenèse des ribosomes

Translation is an essential metabolic activity in all cells, carried by ribosomes. These large complexes are synthetized in the nucleolus, and require the coordinated expression of 4 ribosomal RNA, 80 ribosomal proteins, and more than 200 assembly factors. Indeed, their biogenesis is both complex and expensive, consuming more than half of the energy in proliferating cells. As the cellular need for ribosomes varies with environmental or metabolic conditions, it is no surprise that their synthesis is tightly regulated in response to a number of cues. Many mechanisms ensure that the intensity of ribosome biogenesis is coupled to cell homeostasis. One of them is the ability of ribosomal proteins to regulate gene expression at many levels, ranging from the translation specificity to the activation or repression of transcription. Many of these functions are performed off the ribosome, and are therefore termed extraribosomal. Our team has discovered a new extraribosomal function of ribosomal protein uL11 in Drosophila. Indeed, when it is trimethylated on lysine 3 (uL11K3me3), it associates with Corto, a transcription factor of the Enhancers of Trithorax and Polycomb family. By studying their genome-wide binding profile on chromatin in S2 cells, we show that these proteins are distributed along different patterns, and that uL11K3me3 specifically binds a subset of active genes enriched in ribosome biogenesis components. Additionally, we generated the first genetic alleles for Drosophila uL11 and describe the molecular screening method that we employed. Last, we studied the phenotypes of uL11 alleles that delete or replace lysine 3. We describe that their Minute-like phenotypes suggest an essential role for the N-terminal domain of uL11, but that it may not result from the association between Corto and uL11K3me3.La traduction est une activité métabolique essentielle dans les cellules, réalisée par les ribosomes. Ces particules sont synthetisées dans le nucléole, ce qui nécessite l’expression coordonnée de 4 ARN ribosomaux, 80 protéines ribosomales, et plus de 200 facteurs d’assemblage. En effet, leur biogenèse est complexe et coûteuse, sollicitant plus de la moitié de l’énergie des cellules en prolifération. La quantité de ribosomes requise varie selon les conditions environnementales et métaboliques, et de ce fait, leur synthèse est modulée en réponse à de nombreux stimuli. De nombreux mécanismes assurent la coordination de la biogenèse des ribosomes et de l’homéostasie cellulaire. L’un d’eux est la capacité des protéines ribosomiques à réguler l’expression des gènes à tous les niveaux, depuis la spécificité de la traduction jusqu’à l’activation ou la répression transcriptionnelle des gènes. Nombre de ces fonctions sont effectuées hors du ribosome et sont donc qualifiées d’extraribosomales. Notre équipe a mis en évidence une nouvelle fonction extraribosomale de la protéine ribosomale uL11 chez la Drosophile. En effet, quand sa lysine 3 est triméthylée (uL11K3me3), elle interagit avec Corto, un facteur de transcription de la famille des Enhancers de Trithorax et Polycomb. En étudiant leur fixation à la chromatine, nous avons montré que ces protéines se répartissent différemment à l’échelle du génome, et que uL11K3me3 est présente au niveau d’un sous-ensemble de gènes actifs enrichi en composants de la biogenèse des ribosomes. De plus, nous avons généré les premiers allèles génétiques du gène uL11 chez la Drosophile, et nous décrivons la stratégie de crible moléculaire employée pour leur identification. Finalement, nous avons étudié les phénotypes des mutants de uL11 dont la lysine 3 est délétée ou substituée. Nous décrivons que leurs phénotypes ressemblent à ceux des mutants Minute, et suggèrent que le domaine N-terminal de uL11 possède une fonction essentielle, mais peut-être indépendante de l’association entre uL11K3m3 et Corto

Single amino-acid mutation in a Drosoph ila melanogaster ribosomal protein: An insight in uL11 transcriptional activity.

The ribosomal protein uL11 is located at the basis of the ribosome P-stalk and plays a paramount role in translational efficiency. In addition, no mutant for uL11 is available suggesting that this gene is haplo-insufficient as many other Ribosomal Protein Genes (RPGs). We have previously shown that overexpression of Drosophila melanogaster uL11 enhances the transcription of many RPGs and Ribosomal Biogenesis genes (RiBis) suggesting that uL11 might globally regulate the level of translation through its transcriptional activity. Moreover, uL11 trimethylated on lysine 3 (uL11K3me3) interacts with the chromodomain of the Enhancer of Polycomb and Trithorax Corto, and both proteins co-localize with RNA Polymerase II at many sites on polytene chromosomes. These data have led to the hypothesis that the N-terminal end of uL11, and more particularly the trimethylation of lysine 3, supports the extra-ribosomal activity of uL11 in transcription. To address this question, we mutated the lysine 3 codon using a CRISPR/Cas9 strategy and obtained several lysine 3 mutants. We describe here the first mutants of D. melanogaster uL11. Unexpectedly, the uL11K3A mutant, in which the lysine 3 codon is replaced by an alanine, displays a genuine Minute phenotype known to be characteristic of RPG deletions (longer development, low fertility, high lethality, thin and short bristles) whereas the uL11K3Y mutant, in which the lysine 3 codon is replaced by a tyrosine, is unaffected. In agreement, the rate of translation decreases in uL11K3A but not in uL11K3Y. Co-immunoprecipitation experiments show that the interaction between uL11 and the Corto chromodomain is impaired by both mutations. However, Histone Association Assays indicate that the mutant proteins still bind chromatin. RNA-seq analyses from wing imaginal discs show that Corto represses RPG expression whereas very few genes are deregulated in uL11 mutants. We propose that Corto, by repressing RPG expression, ensures that all ribosomal proteins are present at the correct stoichiometry, and that uL11 fine-tunes its transcriptional regulation of RPGs

Single amino-acid mutation in a Drosoph ila melanogaster ribosomal protein: An insight in uL11 transcriptional activity

International audienceThe ribosomal protein uL11 is located at the basis of the ribosome P-stalk and plays a paramount role in translational efficiency. In addition, no mutant for uL11 is available suggesting that this gene is haplo-insufficient as many other Ribosomal Protein Genes ( RPGs ). We have previously shown that overexpression of Drosophila melanogaster uL11 enhances the transcription of many RPGs and Ribosomal Biogenesis genes ( RiBis ) suggesting that uL11 might globally regulate the level of translation through its transcriptional activity. Moreover, uL11 trimethylated on lysine 3 (uL11K3me3) interacts with the chromodomain of the Enhancer of Polycomb and Trithorax Corto, and both proteins co-localize with RNA Polymerase II at many sites on polytene chromosomes. These data have led to the hypothesis that the N-terminal end of uL11, and more particularly the trimethylation of lysine 3, supports the extra-ribosomal activity of uL11 in transcription. To address this question, we mutated the lysine 3 codon using a CRISPR/Cas9 strategy and obtained several lysine 3 mutants. We describe here the first mutants of D . melanogaster uL11 . Unexpectedly, the uL11 K3A mutant, in which the lysine 3 codon is replaced by an alanine, displays a genuine Minute phenotype known to be characteristic of RPG deletions (longer development, low fertility, high lethality, thin and short bristles) whereas the uL11 K3Y mutant, in which the lysine 3 codon is replaced by a tyrosine, is unaffected. In agreement, the rate of translation decreases in uL11 K3A but not in uL11 K3Y . Co-immunoprecipitation experiments show that the interaction between uL11 and the Corto chromodomain is impaired by both mutations. However, Histone Association Assays indicate that the mutant proteins still bind chromatin. RNA-seq analyses from wing imaginal discs show that Corto represses RPG expression whereas very few genes are deregulated in uL11 mutants. We propose that Corto, by repressing RPG expression, ensures that all ribosomal proteins are present at the correct stoichiometry, and that uL11 fine-tunes its transcriptional regulation of RPGs

Cyclin G and the Polycomb Repressive complexes PRC1 and PR-DUB cooperate for developmental stability

International audienc

<i>CycG</i> interacts with several <i>PcG</i> and <i>ETP</i> genes for developmental stability.

<p>Centroid size FA (FA10) of <i>ETP</i> or <i>PcG</i> heterozygous mutant females combined with <i>da-Gal4</i>, <i>UAS-CycG</i>^<i>ΔP</i> (dark orange; <i>da-Gal4</i>, <i>UAS-CycG</i>^<i>ΔP</i>; <i>PcG/+</i> or <i>da-Gal4</i>, <i>UAS-CycG</i>^<i>ΔP</i>; <i>ETP/+</i>) and <i>ETP</i> or <i>PcG</i> heterozygous mutant females combined with <i>da-Gal4</i> (blue; <i>da-Gal4/+; PcG/+</i> or <i>da-Gal4</i>; <i>ETP/+</i>). The grey dashed line shows FA of <i>da-Gal4</i>, <i>UAS-CycG</i>^<i>ΔP</i><i>/+</i> females. (F-tests, *p-value<0.05; ** p-value<0.01; *** p-value<0.001, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007498#pgen.1007498.s007" target="_blank">S5 Table</a>). Source data are provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007498#pgen.1007498.s008" target="_blank">S6 Table</a>.</p

The ETP interacting domain limits <i>CycG</i>-induced FA.

<p><b>A–</b>Map of the 566 amino-acid Cyclin G protein showing the ETP interacting and PEST domains. <b>B–</b>Wing centroid size FA (FA10) of females <i>da-Gal4</i>/+ (+), <i>+/UAS-CycG</i>^<i>FL</i><i>; da-Gal4/+</i>, (<i>CycG</i>^<i>FL</i>) and <i>+/UAS-CycG</i>^<i>ΔE</i>; <i>da-Gal4</i>, (<i>CycG</i>^<i>ΔE</i>). <b>C–</b>Wing centroid size FA (FA10) of females <i>da-Gal4</i>/+ (+), <i>+/ UAS-CycG</i>^<i>ΔP</i><i>; da-Gal4/+</i> (<i>CycG</i>^<i>ΔP</i>) and <i>+/UAS-CycG</i>^{<i>ΔEΔP</i>}<i>; da-Gal4/+</i> (<i>CycG</i>^{<i>ΔEΔP</i>}). (F-tests, *** p-value<0.001, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007498#pgen.1007498.s005" target="_blank">S3 Table</a>). Source data are provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007498#pgen.1007498.s006" target="_blank">S4 Table</a>.</p

Genes deregulated in wing imaginal discs expressing <i>CycG</i>^<i>ΔP</i>.

<p><b>A–</b>RT-qPCR analysis of endogenous <i>CycG</i> expression in <i>da-Gal4</i>,<i>UAS-CycG</i>^<i>ΔP</i><i>/+</i> and <i>da-Gal4/+</i> wing imaginal discs. Expression of <i>CycG</i> was normalized on the geometric mean of <i>Lam</i> and <i>rin</i> (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007498#pgen.1007498.s010" target="_blank">S8 Table</a>). t-tests, ** p-value<0.01. Error bars correspond to standard deviations. <b>B–</b>Ontology of up-regulated and down-regulated genes in <i>da-Gal4</i>, <i>UAS-CycG</i>^<i>ΔP</i><i>/+ vs da-Gal4/+</i> wing imaginal discs. Gene ontology analysis was performed with DAVID (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007498#pgen.1007498.s011" target="_blank">S9 Table</a>). <b>C–</b>RT-qPCR analysis of <i>RPL15</i>, <i>RPL7</i> and <i>Rack1</i> expression in <i>da-Gal4</i>, <i>UAS-CycG</i>^<i>ΔP</i><i>/+</i> and <i>da-Gal4/+</i> wing imaginal discs. Expression of <i>RPL15</i>, <i>RPL7</i> and <i>Rack1</i> were normalized on the geometric mean of <i>Lam</i> and <i>rin</i> (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007498#pgen.1007498.s012" target="_blank">S10 Table</a>). t-tests, ** p-value<0.01. Error bars correspond to standard deviations. t-tests, ** p-value<0.01; *** p-value<0.001.</p

Snapshots illustrated Cyclin G ChIP-seq.

<p>Focus on <i>RPL7</i> (A), <i>RPL5</i> (B), <i>Rack1</i> (C) and <i>CycG</i> (D).</p

Cyclin G co-localizes with H2AK118ub at many sites on polytene chromosomes but overexpression of <i>CycG</i> does not modify global H2AK118ub.

<p><b>A, A’, A”–</b>Immunostaining of polytene chromosomes from <i>w</i>^<i>1118</i> third instar larvae. H2AK118ub (red), Cyclin G (green), DAPI (blue). <b>A”‘</b>–Close-up of the box showed in A”. <b>B, B’–</b>Wing imaginal discs of 3^rd instar larvae expressing <i>CycG</i>^<i>ΔP</i> in the posterior compartment under control of the <i>en-Gal4</i> driver, stained with anti-Cyclin G (green) and anti-H2AK118ub (red). <b>C, C’–</b>Wing imaginal discs of 3^rd instar larvae expressing <i>CycG</i>^{<i>ΔEΔP</i>} in the posterior compartment under control of the <i>en-Gal4</i> driver, stained with anti-Cyclin G (green) and anti-H2AK118ub (red). <b>D, D’, D”, D”’</b>–GFP clones in wing imaginal discs stained with anti-H2AK118ub (red). D’, D” and D”’ are close-up views of the yellow rectangle shown in D. <b>E, E’, E”, E”’</b>–<i>CycG</i>^<i>ΔP</i> clones marked by GFP in wing imaginal discs stained with anti-H2AK118ub (red). E’, E” and E”’ are close-up views of the yellow rectangle shown in E. <b>F, F’, F”, F”’</b>–<i>CycG</i>^{<i>ΔEΔP</i>} clones marked by GFP in wing imaginal discs stained with anti-H2AK118ub (red). F’, F” and F”’ are close-up views of the yellow rectangle shown in F. Scale bars: 50 μm.</p

Functional subnetwork identified in wing imaginal discs expressing <i>CycG</i>^<i>ΔP</i>.

<p>Schematic representation of a sub-network of 222 genes centred on Cyclin G (CycG_subnetwork.xmml) and identified using JactiveModules (Z score 48.53). In this sub-network, 65 genes were up-regulated in <i>da-Gal4</i>, <i>UAS-CycG</i>^<i>ΔP</i> <i>vs da-Gal4/+</i> wing imaginal discs (green gradient), 124 genes were down-regulated (red gradient), and 33 genes were not significantly deregulated (grey). Genes bound by Cyclin G are circled in blue. Transcription factor genes are represented by squares. Genes were clustered depending on their function. Black edges correspond to interactions discovered in the present study. Grey edges correspond to interactions described in the literature and imported into the WID network using DroID.</p