The cytoplasmic fraction of the histone lysine methyltransferase Setdb1 is essential for embryonic stem cells.

The major lysine methyltransferase (KMT) Setdb1 is essential for self-renewal and viability of mouse embryonic stem cells (mESCs). Setdb1 was primarily known to methylate the lysine 9 of histone 3 (H3K9) in the nucleus, where it regulates chromatin functions. However, Setdb1 is also massively localized in the cytoplasm, including in mESCs, where its role remains elusive. Here, we show that the cytoplasmic Setdb1 (cSetdb1) is essential for the survival of mESCs. Yeast two-hybrid analysis revealed that cSetdb1 interacts with several regulators of mRNA stability and protein translation machinery, such as the ESCs-specific E3 ubiquitin ligase and mRNA silencer Trim71/Lin41. We found that cSetdb1 is required for the integrity of Trim71 complex(es) involved in mRNA metabolism and translation. cSetdb1 modulates the abundance of mRNAs and the rate of newly synthesized proteins. Altogether, our data uncovered the cytoplasmic post-transcriptional regulation of gene expression mediated by a key epigenetic regulator.

Glucose controls co-translation of structurally related mRNAs via the mTOR and eIF2 pathways in human pancreatic beta cells.

Pancreatic beta cell response to glucose is critical for the maintenance of normoglycemia. A strong transcriptional response was classically described in rodent models but, interestingly, not in human cells. In this study, we exposed human pancreatic beta cells to an increased concentration of glucose and analysed at a global level the mRNAs steady state levels and their translationalability. Polysome profiling analysis showed an early acute increase in protein synthesis and a specific translation regulation of more than 400 mRNAs, independently of their transcriptional regulation. We clustered the co-regulated mRNAs according to their behaviour in translation in response to glucose and discovered common structural and sequence mRNA features. Among them mTOR- and eIF2-sensitive elements have a predominant role to increase mostly the translation of mRNAs encoding for proteins of the translational machinery. Furthermore, we show that mTOR and eIF2α pathways are independently regulated in response to glucose, participating to a translational reshaping to adapt beta cell metabolism. The early acute increase in the translation machinery components prepare the beta cell for further protein demand due to glucose-mediated metabolism changes.

Glucose treatment of human pancreatic ß-cells enhances translation of mRNAs involved in energetics and insulin secretion.

Glucose-mediated signaling regulates the expression of a limited number of genes in human pancreatic ß-cells at the transcriptional level. However, it is unclear whether glucose plays a role in posttranscriptional RNA processing or translational control of gene expression. Here, we asked whether glucose affects posttranscriptional steps and regulates protein synthesis in human ß-cell lines. We first showed the involvement of the mTOR pathway in glucose-related signaling. We also used the surface sensing of translation technique, based on puromycin incorporation into newly translated proteins, to demonstrate that glucose treatment increased protein translation. Among the list of glucose-induced proteins, we identified the proconvertase PCSK1, an enzyme involved in the proteolytic conversion of proinsulin to insulin, whose translation was induced within minutes following glucose treatment. We finally performed global proteomic analysis by mass spectrometry to characterize newly translated proteins upon glucose treatment. We found enrichment in proteins involved in translation, glycolysis, TCA metabolism, and insulin secretion. Taken together, our study demonstrates that, although glucose minorly affects gene transcription in human ß-cells, it plays a major role at the translational level.

Integrated analyses of translatome and proteome identify the rules of translation selectivity in RPS14-deficient cells.

In ribosomopathies, the Diamond-Blackfan anemia (DBA) or 5q- syndrome, ribosomal protein (RP) genes are affected by mutation or deletion, resulting in bone marrow erythroid hypoplasia. Unbalanced production of ribosomal subunits leading to a limited ribosome cellular content regulates translation at the expense of the master erythroid transcription factor GATA1. In RPS14-deficient cells mimicking 5q- syndrome erythroid defects, we show that the transcript length, codon bias of the coding sequence (CDS) and 3'UTR (untranslated region) structure are the key determinants of translation. In these cells, short transcripts with a structured 3'UTR and high codon adaptation index (CAI) showed a decreased translation efficiency. Quantitative analysis of the whole proteome confirmed that the post-transcriptional changes depended on the transcript characteristics that governed the translation efficiency in conditions of low ribosome availability. In addition, proteins involved in normal erythroid differentiation share most determinants of translation selectivity. Our findings thus indicate that impaired erythroid maturation due to 5q- syndrome may proceed from a translational selectivity at the expense of the erythroid differentiation program, and suggest that an interplay between the CDS and UTR may regulate mRNA translation.

Conventional and unconventional interactions of the transcription factor FOXL2 uncovered by a proteome-wide analysis.

Beyond the study of its transcriptional target genes, the identification of the various interactors of a transcription factor (TF) is crucial to understand its diverse cellular roles. We focused on FOXL2, a winged-helix forkhead TF important for ovarian development and maintenance. FOXL2 has been implicated in diverse cellular processes, including apoptosis, the control of cell cycle or the regulation of steroid hormone synthesis. To reliably identify partners of endogenous FOXL2, we performed a proteome-wide analysis using co-immunoprecipitation in the murine granulosa cell-derived AT29c and the pituitary-derived alpha-T3 cell lines, using three antibodies targeting different parts of the protein. Following a stringent selection of mass spectrometry data on the basis of identification reliability and protein enrichment, we identified a core set of 255 partners common to both cell lines. Their analysis showed that we could co-precipitate several complexes involved in mRNA processing, chromatin remodeling and DNA replication and repair. We further validated (direct and/or indirect) interactions with selected partners, suggesting an unexpected role for FOXL2 in those processes. Overall, this comprehensive analysis of the endogenous FOXL2 interactome sheds light on its numerous and diverse interactors and unconventional cellular roles.

A SUMO-dependent feedback loop senses and controls the biogenesis of nuclear pore subunits.

While the activity of multiprotein complexes is crucial for cellular metabolism, little is known about the mechanisms that collectively control the expression of their components. Here, we investigate the regulations targeting the biogenesis of the nuclear pore complex (NPC), the macromolecular assembly mediating nucleocytoplasmic exchanges. Systematic analysis of RNA-binding proteins interactomes, together with in vivo and in vitro assays, reveal that a subset of NPC mRNAs are specifically bound by Hek2, a yeast hnRNP K-like protein. Hek2-dependent translational repression and protein turnover are further shown to finely tune the levels of NPC subunits. Strikingly, mutations or physiological perturbations altering pore integrity decrease the levels of the NPC-associated SUMO protease Ulp1, and trigger the accumulation of sumoylated versions of Hek2 unable to bind NPC mRNAs. Our results support the existence of a quality control mechanism involving Ulp1 as a sensor of NPC integrity and Hek2 as a repressor of NPC biogenesis.

IRFinder: assessing the impact of intron retention on mammalian gene expression.

Intron retention (IR) occurs when an intron is transcribed into pre-mRNA and remains in the final mRNA. We have developed a program and database called IRFinder to accurately detect IR from mRNA sequencing data. Analysis of 2573 samples showed that IR occurs in all tissues analyzed, affects over 80% of all coding genes and is associated with cell differentiation and the cell cycle. Frequently retained introns are enriched for specific RNA binding protein sites and are often retained in clusters in the same gene. IR is associated with lower protein levels and intron-retaining transcripts that escape nonsense-mediated decay are not actively translated.

Tracking a refined eIF4E-binding motif reveals Angel1 as a new partner of eIF4E.

The initiation factor 4E (eIF4E) is implicated in most of the crucial steps of the mRNA life cycle and is recognized as a pivotal protein in gene regulation. Many of these roles are mediated by its interaction with specific proteins generally known as eIF4E-interacting partners (4E-IPs), such as eIF4G and 4E-BP. To screen for new 4E-IPs, we developed a novel approach based on structural, in silico and biochemical analyses. We identified the protein Angel1, a member of the CCR4 deadenylase family. Immunoprecipitation experiments provided evidence that Angel1 is able to interact in vitro and in vivo with eIF4E. Point mutation variants of Angel1 demonstrated that the interaction of Angel1 with eIF4E is mediated through a consensus eIF4E-binding motif. Immunofluorescence and cell fractionation experiments showed that Angel1 is confined to the endoplasmic reticulum and Golgi apparatus, where it partially co-localizes with eIF4E and eIF4G, but not with 4E-BP. Furthermore, manipulating Angel1 levels in living cells had no effect on global translation rates, suggesting that the protein has a more specific function. Taken together, our results illustrate that we developed a powerful method for identifying new eIF4E partners and open new perspectives for understanding eIF4E-specific regulation.

Dephosphorylation of eIF2α is essential for protein synthesis increase and cell cycle progression after sea urchin fertilization.

The eukaryotic Initiation Factor 2 (eIF2) is a key regulator of protein synthesis in eukaryotic cells, implicated in the initiation step of translation. Fertilization of the sea urchin eggs triggers a rapid increase in protein synthesis activity, which is necessary for the progress into embryonic cell cycles. Here we demonstrate that fertilization triggers eIF2α dephosphorylation, concomitant with an increase in protein synthesis and that induction of the eIF2α phosphorylation is intimately linked with an inhibition of protein synthesis and cell cycle arrest. Using a phospho-mimetic protein microinjected into sea urchin eggs, we showed that dephosphorylation of eIF2α is necessary for protein synthesis activity and cell division progression following fertilization. Our results demonstrate that regulation of eIF2α plays an important role in the protein synthesis rise that occurs during early development following fertilization.

The translational repressor 4E-BP called to order by eIF4E: new structural insights by SAXS.

eIF4E binding protein (4E-BP) inhibits translation of capped mRNA by binding to the initiation factor eIF4E and is known to be mostly or completely unstructured in both free and bound states. Using small angle X-ray scattering (SAXS), we report here the analysis of 4E-BP structure in solution, which reveals that while 4E-BP is intrinsically disordered in the free state, it undergoes a dramatic compaction in the bound state. Our results demonstrate that 4E-BP and eIF4E form a 'fuzzy complex', challenging current visions of eIF4E/4E-BP complex regulation.

The rotaviral NSP3 protein stimulates translation of polyadenylated target mRNAs independently of its RNA-binding domain.

The non-structural protein 3 (NSP3) of rotaviruses is an RNA-binding protein that specifically recognises a 4 nucleotide sequence at the 3' extremity of the non-polyadenylated viral mRNAs. NSP3 also has a high affinity for eIF4G. These two functions are clearly delimited in separate domains the structures of which have been determined. They are joined by a central domain implicated in the dimerisation of the full length protein. The bridging function of NSP3 between the 3' end of the viral mRNA and eIF4G has been proposed to enhance the synthesis of viral proteins. However, this role has been questioned as knock-down of NSP3 did not impair viral protein synthesis. We show here using a MS2/MS2-CP tethering assay that a C-terminal fragment of NSP3 containing the eIF4G binding domain and the dimerisation domain can increase the expression of a protein encoded by a target reporter mRNA in HEK 293 cells. The amount of reporter mRNA in the cells is not significantly affected by the presence of the NSP3 derived fusion protein showing that the enhanced protein expression is due to increased translation. These results show that NSP3 can act as a translational enhancer even on a polyadenylated mRNA that should be a substrate for PABP1.

A variant mimicking hyperphosphorylated 4E-BP inhibits protein synthesis in a sea urchin cell-free, cap-dependent translation system.

BACKGROUND: 4E-BP is a translational inhibitor that binds to eIF4E to repress cap-dependent translation initiation. This critical protein:protein interaction is regulated by the phosphorylation of 4E-BP. Hypophosphorylated 4E-BP binds to eIF4E and inhibits cap-dependent translation, whereas hyperphosphorylated forms do not. While three 4E-BP proteins exist in mammals, only one gene encoding for 4E-BP is present in the sea urchin genome. The protein product has a highly conserved core domain containing the eIF4E-binding domain motif (YxxxxLPhi) and four of the regulatory phosphorylation sites. METHODOLOGY/PRINCIPAL FINDINGS: Using a sea urchin cell-free cap-dependent translation system prepared from fertilized eggs, we provide the first direct evidence that the sea urchin 4E-BP inhibits cap-dependent translation. We show here that a sea urchin 4E-BP variant, mimicking phosphorylation on four core residues required to abrogate binding to eIF4E, surprisingly maintains physical association to eIF4E and inhibits protein synthesis. CONCLUSIONS/SIGNIFICANCE: Here, we examine the involvement of the evolutionarily conserved core domain and phosphorylation sites of sea urchin 4E-BP in the regulation of eIF4E-binding. These studies primarily demonstrate the conserved activity of the 4E-BP translational repressor and the importance of the eIF4E-binding domain in sea urchin. We also show that a variant mimicking hyperphosphorylation of the four regulatory phosphorylation sites common to sea urchin and human 4E-BP is not sufficient for release from eIF4E and translation promotion. Therefore, our results suggest that there are additional mechanisms to that of phosphorylation at the four critical sites of 4E-BP that are required to disrupt binding to eIF4E.

[Gene expression regulation at the translational level: contribution of marine organisms]. / Régulation de l'expression des gènes au niveau de la traduction: intérêt des modèles marins.

Gene expression regulation is crucial for organism survival. Each step has to be regulated, from the gene to the protein. mRNA can be stored in the cell without any direct translation. This process is used by the cell to control protein synthesis rapidly at the right place, at the right time. Protein synthesis costs a lot of energy for the cell, so that a precise control of this process is required. Translation initiation represents an important step to regulate gene expression. Many factors that can bind mRNA and recruit different partners are involved in the inhibition or stimulation of protein synthesis. Oceans contain an important diversity of organisms that are used as important models to analyse gene expression at the translational level. These are useful to study translational control in different physiological processes for instance cell cycle (meiosis during meiotic maturation of starfish oocytes, mitosis following fertilization of sea urchin eggs) or to understand nervous system mechanisms (aplysia). All these studies will help finding novel actors involved in translational control and will thus be useful to discover new targets for therapeutic treatments against human diseases.

[Initiation factors eIF4: from sea urchin embryonic development to chronic lymphocytic leukemia]. / Facteurs d'initiation eIF4: du développement embryonnaire de l'oursin à la leucémie lymphoïde chronique.

mRNA translation is now recognized as a important regulatory step for gene expression in different physiological and pathophysiological processes including cell proliferation and apoptosis. B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of resting lymphocytes and defective apoptosis. The mRNA cap-binding protein eIF4E (eukaryotic Initiation Factor 4E) and its repressor 4E-BP (eIF4E Binding protein) are crucial translational regulators that have been involved in survival and apoptosis processes of cells. We have shown that the release of eIF4E from its translational repressor 4E-BP is an important event for the first mitotic division triggered by fertilization and that the degradation of 4E-BP is a new means to regulate 4E-BP function that has to be analyzed in other physiological and physiopathological processes. In this chapter, we describe recent advances illustrating the importance of eIF4E and 4E-BP in cancer processes, suggesting that these actors can be targeted for potential therapy against cancer in general and LLC in particular.

[Sea urchin embryo, DNA-damaged cell cycle checkpoint and the mechanisms initiating cancer development]. / L'embryon d'oursin, le point de surveillance de l'ADN endommagé de la division cellulaire et les mécanismes à l'origine de la cancérisation.

Cell division is an essential process for heredity, maintenance and evolution of the whole living kingdom. Sea urchin early development represents an excellent experimental model for the analysis of cell cycle checkpoint mechanisms since embryonic cells contain a functional DNA-damage checkpoint and since the whole sea urchin genome is sequenced. The DNA-damaged checkpoint is responsible for an arrest in the cell cycle when DNA is damaged or incorrectly replicated, for activation of the DNA repair mechanism, and for commitment to cell death by apoptosis in the case of failure to repair. New insights in cancer biology lead to two fundamental concepts about the very first origin of cancerogenesis. Cancers result from dysfunction of DNA-damaged checkpoints and cancers appear as a result of normal stem cell (NCS) transformation into a cancer stem cell (CSC). The second aspect suggests a new definition of "cancer", since CSC can be detected well before any clinical evidence. Since early development starts from the zygote, which is a primary stem cell, sea urchin early development allows analysis of the early steps of the cancerization process. Although sea urchins do not develop cancers, the model is alternative and complementary to stem cells which are not easy to isolate, do not divide in a short time and do not divide synchronously. In the field of toxicology and incidence on human health, the sea urchin experimental model allows assessment of cancer risk from single or combined molecules long before any epidemiologic evidence is available. Sea urchin embryos were used to test the worldwide used pesticide Roundup that contains glyphosate as the active herbicide agent; it was shown to activate the DNA-damage checkpoint of the first cell cycle of development. The model therefore allows considerable increase in risk evaluation of new products in the field of cancer and offers a tool for the discovery of molecular markers for early diagnostic in cancer biology. Prevention and early diagnosis are two decisive elements of human cancer therapy.

After fertilization of sea urchin eggs, eIF4G is post-translationally modified and associated with the cap-binding protein eIF4E.

Release of eukaryotic initiation factor 4E (eIF4E) from its translational repressor eIF4E-binding protein (4E-BP) is a crucial event for the first mitotic division following fertilization of sea urchin eggs. Finding partners of eIF4E following fertilization is crucial to understand how eIF4E functions during this physiological process. The isolation and characterization of cDNA encoding Sphaerechinus granularis eIF4G (SgIF4G) are reported. mRNA of SgIF4G is present as a single 8.5-kb transcript in unfertilized eggs, suggesting that only one ortholog exists in echinoderms. The longest open reading frame predicts a sequence of 5235 nucleotides encoding a deduced polypeptide of 1745 amino acids with a predicted molecular mass of 192 kDa. Among highly conserved domains, SgIF4G protein possesses motifs that correspond to the poly(A) binding protein and eIF4E protein-binding sites. A specific polyclonal antibody was produced and used to characterize the SgIF4G protein in unfertilized and fertilized eggs by SDS-PAGE and western blotting. Multiple differentially migrating bands representing isoforms of sea urchin eIF4G are present in unfertilized eggs. Fertilization triggers modifications of the SgIF4G isoforms and rapid formation of the SgIF4G-eIF4E complex. Whereas rapamycin inhibits the formation of the SgIF4G-eIF4E complex, modification of these SgIF4G isoforms occurs independently from the rapamycin-sensitive pathway. Microinjection of a peptide corresponding to the eIF4E-binding site derived from the sequence of SgIF4G into unfertilized eggs affects the first mitotic division of sea urchin embryos. Association of SgIF4G with eIF4E is a crucial event for the onset of the first mitotic division following fertilization, suggesting that cap-dependent translation is highly regulated during this process. This hypothesis is strengthened by the evidence that microinjection of the cap analog m(7)GDP into unfertilized eggs inhibits the first mitotic division.

The genomic repertoire for cell cycle control and DNA metabolism in S. purpuratus.

A search of the Strongylocentrotus purpuratus genome for genes associated with cell cycle control and DNA metabolism shows that the known repertoire of these genes is conserved in the sea urchin, although with fewer family members represented than in vertebrates, and with some cases of echinoderm-specific gene diversifications. For example, while homologues of the known cyclins are mostly encoded by single genes in S. purpuratus (unlike vertebrates, which have multiple isoforms), there are additional genes encoding novel cyclins of the B and K/L types. Almost all known cyclin-dependent kinases (CDKs) or CDK-like proteins have an orthologue in S. purpuratus; CDK3 is one exception, whereas CDK4 and 6 are represented by a single homologue, referred to as CDK4. While the complexity of the two families of mitotic kinases, Polo and Aurora, is close to that found in the nematode, the diversity of the NIMA-related kinases (NEK proteins) approaches that of vertebrates. Among the nine NEK proteins found in S. purpuratus, eight could be assigned orthologues in vertebrates, whereas the ninth is unique to sea urchins. Most known DNA replication, DNA repair and mitotic checkpoint genes are also present, as are homologues of the pRB (two) and p53 (one) tumor suppressors. Interestingly, the p21/p27 family of CDK inhibitors is represented by one homologue, whereas the INK4 and ARF families of tumor suppressors appear to be absent, suggesting that these evolved only in vertebrates. Our results suggest that, while the cell cycle control mechanisms known from other animals are generally conserved in sea urchin, parts of the machinery have diversified within the echinoderm lineage. The set of genes uncovered in this analysis of the S. purpuratus genome should enhance future research on cell cycle control and developmental regulation in this model.

Translational control genes in the sea urchin genome.

Sea urchin eggs and early cleavage stage embryos provide an example of regulated gene expression at the level of translation. The availability of the sea urchin genome offers the opportunity to investigate the "translational control" toolkit of this model system. The annotation of the genome reveals that most of the factors implicated in translational control are encoded by nonredundant genes in echinoderm, an advantage for future functional studies. In this paper, we focus on translation factors that have been shown or suggested to play crucial role in cell cycle and development of sea urchin embryos. Addressing the cap-binding translational control, three closely related eIF4E genes (class I, II, III) are present, whereas its repressor 4E-BP and its activator eIF4G are both encoded by one gene. Analysis of the class III eIF4E proteins in various phyla shows an echinoderm-specific amino acid substitution. Furthermore, an interaction site between eIF4G and poly(A)-binding protein is uncovered in the sea urchin eIF4G proteins and is conserved in metazoan evolution. In silico screening of the sea urchin genome has uncovered potential new regulators of eIF4E sharing the common eIF4E recognition motif. Taking together, these data provide new insights regarding the strong requirement of cap-dependent translation following fertilization. The genome analysis gives insights on the complexity of eEF1B structure and motifs of functional relevance, involved in the translational control of gene expression at the level of elongation. Finally, because deregulation of translation process can lead to diseases and tumor formation in humans, the sea urchin orthologs of human genes implicated in human diseases and signaling pathways regulating translation were also discussed.

CUG-BP1/CELF1 requires UGU-rich sequences for high-affinity binding.

CUG-BP1 [CUG-binding protein 1 also called CELF (CUG-BP1 and ETR3 like factors) 1] is a human RNA-binding protein that has been implicated in the control of splicing and mRNA translation. The Xenopus homologue [EDEN-BP (embryo deadenylation element-binding protein)] is required for rapid deadenylation of certain maternal mRNAs just after fertilization. A variety of sequence elements have been described as target sites for these two proteins but their binding specificity is still controversial. Using a SELEX (systematic evolution of ligand by exponential enrichment) procedure and recombinant CUG-BP1 we selected two families of aptamers. Surface plasmon resonance and electrophoretic mobility-shift assays showed that these two families differed in their ability to bind CUG-BP1. Furthermore, the selected high-affinity aptamers form two complexes with CUG-BP1 in electrophoretic mobility assays whereas those that bind with low affinity only form one complex. The validity of the distinction between the two families of aptamers was confirmed by a functional in vivo deadenylation assay. Only those aptamers that bound CUG-BP1 with high affinity conferred deadenylation on a reporter mRNA. These high-affinity RNAs are characterized by a richness in UGU motifs. Using these binding site characteristics we identified the Xenopus maternal mRNA encoding the MAPK (mitogen-activated protein kinase) phosphatase (XCl100alpha) as a substrate for EDEN-BP. In conclusion, high-affinity CUG-BP1 binding sites are sequence elements at least 30 nucleotides in length that are enriched in combinations of U and G nucleotides and contain at least 4 UGU trinucleotide motifs. Such sequence elements are functionally competent to target an RNA for deadenylation in vivo.

Oligomerization of EDEN-BP is required for specific mRNA deadenylation and binding.

BACKGROUND INFORMATION: mRNA deadenylation [shortening of the poly(A) tail] is often triggered by specific sequence elements present within mRNA 3' untranslated regions and generally causes rapid degradation of the mRNA. In vertebrates, many of these deadenylation elements are called AREs (AU-rich elements). The EDEN (embryo deadenylation element) sequence is a Xenopus class III ARE. EDEN acts by binding a specific factor, EDEN-BP (EDEN-binding protein), which in turn stimulates deadenylation. RESULTS: We show here that EDEN-BP is able to oligomerize. A 27-amino-acid region of EDEN-BP was identified as a key domain for oligomerization. A mutant of EDEN-BP lacking this region was unable to oligomerize, and a peptide corresponding to this region competitively inhibited the oligomerization of full-length EDEN-BP. Impairing oligomerization by either of these two methods specifically abolished EDEN-dependent deadenylation. Furthermore, impairing oligomerization inhibited the binding of EDEN-BP to its target RNA, demonstrating a strong coupling between EDEN-BP oligomerization and RNA binding. CONCLUSIONS: These data, showing that the oligomerization of EDEN-BP is required for binding of the protein on its target RNA and for EDEN-dependent deadenylation in Xenopus embryos, will be important for the identification of cofactors required for the deadenylation process.