Elisa Pesce

Long non-coding antisense RNA controls Uchl1 translation through an embedded SINEB2 repeat

Most of the mammalian genome is transcribed. This generates a vast repertoire of transcripts that includes protein-coding messenger RNAs, long non-coding RNAs (lncRNAs) and repetitive sequences, such as SINEs (short interspersed nuclear elements). A large percentage of ncRNAs are nuclear-enriched with unknown function. Antisense lncRNAs may form sense–antisense pairs by pairing with a protein-coding gene on the opposite strand to regulate epigenetic silencing, transcription and mRNA stability. Here we identify a nuclear-enriched lncRNA antisense to mouse ubiquitin carboxy-terminal hydrolase L1 (Uchl1), a gene involved in brain function and neurodegenerative diseases. Antisense Uchl1 increases UCHL1 protein synthesis at a post-transcriptional level, hereby identifying a new functional class of lncRNAs. Antisense Uchl1 activity depends on the presence of a 5′ overlapping sequence and an embedded inverted SINEB2 element. These features are shared by other natural antisense transcripts and can confer regulatory activity to an artificial antisense to green fluorescent protein. Antisense Uchl1 function is under the control of stress signalling pathways, as mTORC1 inhibition by rapamycin causes an increase in UCHL1 protein that is associated to the shuttling of antisense Uchl1 RNA from the nucleus to the cytoplasm. Antisense Uchl1 RNA is then required for the association of the overlapping sense protein-coding mRNA to active polysomes for translation. These data reveal another layer of gene expression control at the post-transcriptional level.

RACK1 depletion in a mouse model causes lethality, pigmentation deficits and reduction in protein synthesis efficiency

The receptor for activated C-kinase 1 (RACK1) is a conserved structural protein of 40S ribosomes. Strikingly, deletion of RACK1 in yeast homolog Asc1 is not lethal. Mammalian RACK1 also interacts with many nonribosomal proteins, hinting at several extraribosomal functions. A knockout mouse for RACK1 has not previously been described. We produced the first RACK1 mutant mouse, in which both alleles of RACK1 gene are defective in RACK1 expression (ΔF/ΔF), in a pure C57 Black/6 background. In a sample of 287 pups, we observed no ΔF/ΔF mice (72 expected). Dissection and genotyping of embryos at various stages showed that lethality occurs at gastrulation. Heterozygotes (ΔF/+) have skin pigmentation defects with a white belly spot and hypopigmented tail and paws. ΔF/+ have a transient growth deficit (shown by measuring pup size at P11). The pigmentation deficit is partly reverted by p53 deletion, whereas the lethality is not. ΔF/+ livers have mild accumulation of inactive 80S ribosomal subunits by polysomal profile analysis. In ΔF/+ fibroblasts, protein synthesis response to extracellular and pharmacological stimuli is reduced. These results highlight the role of RACK1 as a ribosomal protein converging signaling to the translational apparatus.

SBDS-Deficient Cells Have an Altered Homeostatic Equilibrium due to Translational Inefficiency Which Explains their Reduced Fitness and Provides a Logical Framework for Intervention

Ribosomopathies are a family of inherited disorders caused by mutations in genes necessary for ribosomal function. Shwachman-Diamond Bodian Syndrome (SDS) is an autosomal recessive disease caused, in most patients, by mutations of the SBDS gene. SBDS is a protein required for the maturation of 60S ribosomes. SDS patients present exocrine pancreatic insufficiency, neutropenia, chronic infections, and skeletal abnormalities. Later in life, patients are prone to myelodisplastic syndrome and acute myeloid leukemia (AML). It is unknown why patients develop AML and which cellular alterations are directly due to the loss of the SBDS protein. Here we derived mouse embryonic fibroblast lines from an SbdsR126T/R126T mouse model. After their immortalization, we reconstituted them by adding wild type Sbds. We then performed a comprehensive analysis of cellular functions including colony formation, translational and transcriptional RNA-seq, stress and drug sensitivity. We show that: 1. Mutant Sbds causes a reduction in cellular clonogenic capability and oncogene-induced transformation. 2. Mutant Sbds causes a marked increase in immature 60S subunits, limited impact on mRNA specific initiation of translation, but reduced global protein synthesis capability. 3. Chronic loss of SBDS activity leads to a rewiring of gene expression with reduced ribosomal capability, but increased lysosomal and catabolic activity. 4. Consistently with the gene signature, we found that SBDS loss causes a reduction in ATP and lactate levels, and increased susceptibility to DNA damage. Combining our data, we conclude that a cell-specific fragile phenotype occurs when SBDS protein drops below a threshold level, and propose a new interpretation of the disease.

Sensitivity of Global Translation to mTOR Inhibition in REN Cells Depends on the Equilibrium between eIF4E and 4E-BP1

Initiation is the rate-limiting phase of protein synthesis, controlled by signaling pathways regulating the phosphorylation of translation factors. Initiation has three steps, 43S, 48S and 80S formation. 43S formation is repressed by eIF2α phosphorylation. The subsequent steps, 48S and 80S formation are enabled by growth factors. 48S relies on eIF4E-mediated assembly of eIF4F complex; 4E-BPs competitively displace eIF4E from eIF4F. Two pathways control eIF4F: 1) mTORc1 phosphorylates and inactivates 4E-BPs, leading to eIF4F formation; 2) the Ras-Mnk cascade phosphorylates eIF4E. We show that REN and NCI-H28 mesothelioma cells have constitutive activation of both pathways and maximal translation rate, in the absence of exogenous growth factors. Translation is rapidly abrogated by phosphorylation of eIF2α. Surprisingly, pharmacological inhibition of mTORc1 leads to the complete dephosphorylation of downstream targets, without changes in methionine incorporation. In addition, the combined administration of mTORc1 and MAPK/Mnk inhibitors has no additive effect. The inhibition of both mTORc1 and mTORc2 does not affect the metabolic rate. In spite of this, mTORc1 inhibition reduces eIF4F complex formation, and depresses translocation of TOP mRNAs on polysomes. Downregulation of eIF4E and overexpression of 4E-BP1 induce rapamycin sensitivity, suggesting that disruption of eIF4F complex, due to eIF4E modulation, competes with its recycling to ribosomes. These data suggest the existence of a dynamic equilibrium in which eIF4F is not essential for all mRNAs and is not displaced from translated mRNAs, before recycling to the next.

Expression and activity of eIF6 trigger Malignant Pleural Mesothelioma growth in vivo

eIF6 is an antiassociation factor that regulates the availability of active 80S. Its activation is driven by the RACK1/PKCβ axis, in a mTORc1 independent manner. We previously described that eIF6 haploinsufficiency causes a striking survival in the Eμ-Myc mouse lymphoma model, with lifespans extended up to 18 months. Here we screen for eIF6 expression in human cancers. We show that Malignant Pleural Mesothelioma tumors (MPM) and a MPM cell line (REN cells) contain high levels of hyperphosphorylated eIF6. Enzastaurin is a PKC beta inhibitor used in clinical trials. We prove that Enzastaurin treatment decreases eIF6 phosphorylation rate, but not eIF6 protein stability. The growth of REN, in vivo, and metastasis are reduced by either Enzastaurin treatment or eIF6 shRNA. Molecular analysis reveals that eIF6 manipulation affects the metabolic status of malignant mesothelioma cells. Less glycolysis and less ATP content are evident in REN cells depleted for eIF6 or treated with Enzastaurin (Anti-Warburg effect). We propose that eIF6 is necessary for malignant mesothelioma growth, in vivo, and can be targeted by kinase inhibitors.

Direct and high throughput (HT) interactions on the ribosomal surface by iRIA

Ribosomes function as platforms for binding of other molecules, but technologies for studying this process are lacking. Therefore we developed iRIA (in vitro Ribosomes Interaction Assay). In approach I, Artemia salina ribosomes spotted on solid phase are used for binding picomoles of analytes; in approach II, cellular extracts allow the measurement of ribosome activity in different conditions. We apply the method to analyze several features of eIF6 binding to 60S subunits. By approach I, we show that the off-rate of eIF6 from preribosomes is slower than from mature ribosomes and that its binding to mature 60S occurs in the nM affinity range. By approach II we show that eIF6 binding sites on 60S are increased with mild eIF6 depletion and decreased in cells that are devoid of SBDS, a ribosomal factor necessary for 60S maturation and involved in Swachman Diamond syndrome. We show binding conditions to immobilized ribosomes adaptable to HT and quantify free ribosomes in cell extracts. In conclusion, we suggest that iRIA will greatly facilitate the study of interactions on the ribosomal surface.

The eukaryotic Initiation Factor 6 (eIF6) regulates ecdysone biosynthesis by modulating translation in Drosophila

During development, ribosome biogenesis and translation reach peak activities, due to impetuous cell proliferation. Current models predict that protein synthesis elevation is controlled by transcription factors and signalling pathways. Developmental models addressing translation factors overexpression effects are lacking. Eukaryotic Initiation Factor (eIF6) is necessary for ribosome biogenesis and efficient translation. eIF6 is a single gene, conserved from yeasts to mammals, suggesting a tight regulation need. We generated a Drosophila melanogaster in vivo model of eIF6 upregulation, demonstrating a boost in general translation and the shut off of the ecdysone biosynthetic pathway. Translation modulation in S2 cells showed that translational rate and ecdysone biosynthesis are inversely correlated. In vivo, eIF6-driven alterations delayed programmed cell death (PCD), resulting in aberrant phenotypes, partially rescued by ecdysone administration. Our data show that eIF6 triggers a translation program with far-reaching effects on metabolism and development, stressing the driving and central role of translation.Translation factors downregulation modulates gene expression but the effect of their overexpression is still unknown. The Eukaryotic Initiation Factor 6 (eIF6) is necessary for ribosome biogenesis and translation initiation. The eif6 gene is a single genetic locus highly conserved from yeast to humans indicating a tight regulation of its gene dosage. eIF6 haploinsufficiency protects mice from lymphomagenesis, and eIF6 is upregulated or amplified in some cancers, but a mechanistic study on the effects of eIF6 overexpression is still lacking. Taking advantage of genetic tractability of D. melanogaster, we characterized the first in vivo model of eIF6 upregulation. Drosophila eIF6 overexpression increases translation and results in a rough eye phenotype due to aberrant apoptosis. Mechanistically, eIF6 reshapes transcription and histone acetylation, disrupting the ecdysone network. This work is the first evidence of how increased translation generates a full transcriptional and hormonal dysregulation, providing new perspectives on the physiological relevance of the translational machinery in regulating gene expression and a model to screen drugs potentially useful to treat cells with altered eif6 gene dosage.Increases in ribosomal proteins and initiation factors are often observed in tumours and required during development. Haploinsufficient models have shown that such elevation is essential for tumour growth. Models with increased gene dosage of initiation factors, addressing the effects of their forced overexpression are lacking. The eukaryotic Initiation Factor 6 (eIF6) gene is amplified in some cancers and overexpressed in most, while it has been demonstrated that eIF6 haploinsufficiency protects mice from lymphomagenesis. eIF6 is necessary for ribosome biogenesis and efficient translation, and is present as a single gene in all animal species. Taking advantage of genetic tractability of Drosophila melanogaster, we generated an in vivo model of eIF6 upregulation, in order to assess the early effects of increased gene dosage of this initiation factor. eIF6 overexpression increases the general rate of translation, both in vivo and in vitro. Organ specific overexpression in the eye causes a rough phenotype. The increase of translation driven by eIF6 is accompanied by a complex transcriptional rewiring and a modulation of histone acetylation activity. Gene expression changes caused by eIF6 include a dominant upregulation of ribosome biogenesis, a shift in Programmed Cell Death (PCD) and inhibition of ecdysteroids biosynthesis. Administration of 20-HydroxyEcdysone or expression of p35 apoptotic modulator reverts some of the effects driven by high eIF6 levels. We conclude that the increased translation driven by high levels of eIF6 generates a transcriptional and hormonal rewiring that evidences the capability of the translational machinery to regulate specific gene expression and metabolism. In addition, our in vivo model could be useful to screen potential drugs to treat cells with altered eIF6 gene dosage.

Increasing Eukaryotic Initiation Factor 6 (eIF6) Gene Dosage Stimulates Global Translation and Induces a Transcriptional and Metabolic Rewiring that Blocks Programmed Cell Death

During development, ribosome biogenesis and translation reach peak activities, due to impetuous cell proliferation. Current models predict that protein synthesis elevation is controlled by transcription factors and signalling pathways. Developmental models addressing translation factors overexpression effects are lacking. Eukaryotic Initiation Factor (eIF6) is necessary for ribosome biogenesis and efficient translation. eIF6 is a single gene, conserved from yeasts to mammals, suggesting a tight regulation need. We generated a Drosophila melanogaster in vivo model of eIF6 upregulation, demonstrating a boost in general translation and the shut off of the ecdysone biosynthetic pathway. Translation modulation in S2 cells showed that translational rate and ecdysone biosynthesis are inversely correlated. In vivo, eIF6-driven alterations delayed programmed cell death (PCD), resulting in aberrant phenotypes, partially rescued by ecdysone administration. Our data show that eIF6 triggers a translation program with far-reaching effects on metabolism and development, stressing the driving and central role of translation.Translation factors downregulation modulates gene expression but the effect of their overexpression is still unknown. The Eukaryotic Initiation Factor 6 (eIF6) is necessary for ribosome biogenesis and translation initiation. The eif6 gene is a single genetic locus highly conserved from yeast to humans indicating a tight regulation of its gene dosage. eIF6 haploinsufficiency protects mice from lymphomagenesis, and eIF6 is upregulated or amplified in some cancers, but a mechanistic study on the effects of eIF6 overexpression is still lacking. Taking advantage of genetic tractability of D. melanogaster, we characterized the first in vivo model of eIF6 upregulation. Drosophila eIF6 overexpression increases translation and results in a rough eye phenotype due to aberrant apoptosis. Mechanistically, eIF6 reshapes transcription and histone acetylation, disrupting the ecdysone network. This work is the first evidence of how increased translation generates a full transcriptional and hormonal dysregulation, providing new perspectives on the physiological relevance of the translational machinery in regulating gene expression and a model to screen drugs potentially useful to treat cells with altered eif6 gene dosage.Increases in ribosomal proteins and initiation factors are often observed in tumours and required during development. Haploinsufficient models have shown that such elevation is essential for tumour growth. Models with increased gene dosage of initiation factors, addressing the effects of their forced overexpression are lacking. The eukaryotic Initiation Factor 6 (eIF6) gene is amplified in some cancers and overexpressed in most, while it has been demonstrated that eIF6 haploinsufficiency protects mice from lymphomagenesis. eIF6 is necessary for ribosome biogenesis and efficient translation, and is present as a single gene in all animal species. Taking advantage of genetic tractability of Drosophila melanogaster, we generated an in vivo model of eIF6 upregulation, in order to assess the early effects of increased gene dosage of this initiation factor. eIF6 overexpression increases the general rate of translation, both in vivo and in vitro. Organ specific overexpression in the eye causes a rough phenotype. The increase of translation driven by eIF6 is accompanied by a complex transcriptional rewiring and a modulation of histone acetylation activity. Gene expression changes caused by eIF6 include a dominant upregulation of ribosome biogenesis, a shift in Programmed Cell Death (PCD) and inhibition of ecdysteroids biosynthesis. Administration of 20-HydroxyEcdysone or expression of p35 apoptotic modulator reverts some of the effects driven by high eIF6 levels. We conclude that the increased translation driven by high levels of eIF6 generates a transcriptional and hormonal rewiring that evidences the capability of the translational machinery to regulate specific gene expression and metabolism. In addition, our in vivo model could be useful to screen potential drugs to treat cells with altered eIF6 gene dosage.

The Drosophila Eukaryotic Initiation Factor eIF6 affects development by regulating apoptosis via the ecdysone pathway

During development, ribosome biogenesis and translation reach peak activities, due to impetuous cell proliferation. Current models predict that protein synthesis elevation is controlled by transcription factors and signalling pathways. Developmental models addressing translation factors overexpression effects are lacking. Eukaryotic Initiation Factor (eIF6) is necessary for ribosome biogenesis and efficient translation. eIF6 is a single gene, conserved from yeasts to mammals, suggesting a tight regulation need. We generated a Drosophila melanogaster in vivo model of eIF6 upregulation, demonstrating a boost in general translation and the shut off of the ecdysone biosynthetic pathway. Translation modulation in S2 cells showed that translational rate and ecdysone biosynthesis are inversely correlated. In vivo, eIF6-driven alterations delayed programmed cell death (PCD), resulting in aberrant phenotypes, partially rescued by ecdysone administration. Our data show that eIF6 triggers a translation program with far-reaching effects on metabolism and development, stressing the driving and central role of translation.Translation factors downregulation modulates gene expression but the effect of their overexpression is still unknown. The Eukaryotic Initiation Factor 6 (eIF6) is necessary for ribosome biogenesis and translation initiation. The eif6 gene is a single genetic locus highly conserved from yeast to humans indicating a tight regulation of its gene dosage. eIF6 haploinsufficiency protects mice from lymphomagenesis, and eIF6 is upregulated or amplified in some cancers, but a mechanistic study on the effects of eIF6 overexpression is still lacking. Taking advantage of genetic tractability of D. melanogaster, we characterized the first in vivo model of eIF6 upregulation. Drosophila eIF6 overexpression increases translation and results in a rough eye phenotype due to aberrant apoptosis. Mechanistically, eIF6 reshapes transcription and histone acetylation, disrupting the ecdysone network. This work is the first evidence of how increased translation generates a full transcriptional and hormonal dysregulation, providing new perspectives on the physiological relevance of the translational machinery in regulating gene expression and a model to screen drugs potentially useful to treat cells with altered eif6 gene dosage.Increases in ribosomal proteins and initiation factors are often observed in tumours and required during development. Haploinsufficient models have shown that such elevation is essential for tumour growth. Models with increased gene dosage of initiation factors, addressing the effects of their forced overexpression are lacking. The eukaryotic Initiation Factor 6 (eIF6) gene is amplified in some cancers and overexpressed in most, while it has been demonstrated that eIF6 haploinsufficiency protects mice from lymphomagenesis. eIF6 is necessary for ribosome biogenesis and efficient translation, and is present as a single gene in all animal species. Taking advantage of genetic tractability of Drosophila melanogaster, we generated an in vivo model of eIF6 upregulation, in order to assess the early effects of increased gene dosage of this initiation factor. eIF6 overexpression increases the general rate of translation, both in vivo and in vitro. Organ specific overexpression in the eye causes a rough phenotype. The increase of translation driven by eIF6 is accompanied by a complex transcriptional rewiring and a modulation of histone acetylation activity. Gene expression changes caused by eIF6 include a dominant upregulation of ribosome biogenesis, a shift in Programmed Cell Death (PCD) and inhibition of ecdysteroids biosynthesis. Administration of 20-HydroxyEcdysone or expression of p35 apoptotic modulator reverts some of the effects driven by high eIF6 levels. We conclude that the increased translation driven by high levels of eIF6 generates a transcriptional and hormonal rewiring that evidences the capability of the translational machinery to regulate specific gene expression and metabolism. In addition, our in vivo model could be useful to screen potential drugs to treat cells with altered eIF6 gene dosage.