Alternative splicing of BCL-x is controlled by RBM25 binding to a G-quadruplex in BCL-x pre-mRNA.

BCL-x is a master regulator of apoptosis whose pre-mRNA is alternatively spliced into either a long (canonical) anti-apoptotic Bcl-xL isoform, or a short (alternative) pro-apoptotic Bcl-xS isoform. The balance between these two antagonistic isoforms is tightly regulated and overexpression of Bcl-xL has been linked to resistance to chemotherapy in several cancers, whereas overexpression of Bcl-xS is associated to some forms of diabetes and cardiac disorders. The splicing factor RBM25 controls alternative splicing of BCL-x: its overexpression favours the production of Bcl-xS, whereas its downregulation has the opposite effect. Here we show that RBM25 directly and specifically binds to GQ-2, an RNA G-quadruplex (rG4) of BCL-x pre-mRNA that forms at the vicinity of the alternative 5' splice site leading to the alternative Bcl-xS isoform. This RBM25/rG4 interaction is crucial for the production of Bcl-xS and depends on the RE (arginine-glutamate-rich) motif of RBM25, thus defining a new type of rG4-interacting domain. PhenDC3, a benchmark G4 ligand, enhances the binding of RBM25 to the GQ-2 rG4 of BCL-x pre-mRNA, thereby promoting the alternative pro-apoptotic Bcl-xS isoform and triggering apoptosis. Furthermore, the screening of a combinatorial library of 90 putative G4 ligands led to the identification of two original compounds, PhenDH8 and PhenDH9, superior to PhenDC3 in promoting the Bcl-xS isoform and apoptosis. Thus, favouring the interaction between RBM25 and the GQ-2 rG4 of BCL-x pre-mRNA represents a relevant intervention point to re-sensitize cancer cells to chemotherapy.

Type I arginine methyltransferases are intervention points to unveil the oncogenic Epstein-Barr virus to the immune system.

The oncogenic Epstein-Barr virus (EBV) evades the immune system but has an Achilles heel: its genome maintenance protein EBNA1. Indeed, EBNA1 is essential for viral genome maintenance but is also highly antigenic. Hence, EBV seemingly evolved a system in which the glycine-alanine repeat (GAr) of EBNA1 limits the translation of its own mRNA to the minimal level to ensure its essential function, thereby, at the same time, minimizing immune recognition. Therefore, defining intervention points at which to interfere with GAr-based inhibition of translation is an important step to trigger an immune response against EBV-carrying cancers. The host protein nucleolin (NCL) plays a critical role in this process via a direct interaction with G-quadruplexes (G4) formed in the GAr-encoding sequence of the viral EBNA1 mRNA. Here we show that the C-terminal arginine-glycine-rich (RGG) motif of NCL is crucial for its role in GAr-based inhibition of translation by mediating interaction of NCL with G4 of EBNA1 mRNA. We also show that this interaction depends on the type I arginine methyltransferase family, notably PRMT1 and PRMT3: drugs or small interfering RNA that target these enzymes prevent efficient binding of NCL on G4 of EBNA1 mRNA and relieve GAr-based inhibition of translation and of antigen presentation. Hence, this work defines type I arginine methyltransferases as therapeutic targets to interfere with EBNA1 and EBV immune evasion.

The nascent polypeptide-associated complex (NAC) controls translation initiation in cis by recruiting nucleolin to the encoding mRNA.

Protein aggregates and abnormal proteins are toxic and associated with neurodegenerative diseases. There are several mechanisms to help cells get rid of aggregates but little is known on how cells prevent aggregate-prone proteins from being synthesised. The EBNA1 of the Epstein-Barr virus (EBV) evades the immune system by suppressing its own mRNA translation initiation in order to minimize the production of antigenic peptides for the major histocompatibility (MHC) class I pathway. Here we show that the emerging peptide of the disordered glycine-alanine repeat (GAr) within EBNA1 dislodges the nascent polypeptide-associated complex (NAC) from the ribosome. This results in the recruitment of nucleolin to the GAr-encoding mRNA and suppression of mRNA translation initiation in cis. Suppressing NAC alpha (NACA) expression prevents nucleolin from binding to the GAr mRNA and overcomes GAr-mediated translation inhibition. Taken together, these observations suggest that EBNA1 exploits a nascent protein quality control pathway to regulate its own rate of synthesis that is based on sensing the nascent GAr peptide by NAC followed by the recruitment of nucleolin to the GAr-encoding RNA sequence.

A homozygous splice variant in ATP5PO, disrupts mitochondrial complex V function and causes Leigh syndrome in two unrelated families.

Mitochondrial complex V plays an important role in oxidative phosphorylation by catalyzing the generation of ATP. Most complex V subunits are nuclear encoded and not yet associated with recognized Mendelian disorders. Using exome sequencing, we identified a rare homozygous splice variant (c.87+3A>G) in ATP5PO, the complex V subunit which encodes the oligomycin sensitivity conferring protein, in three individuals from two unrelated families, with clinical suspicion of a mitochondrial disorder. These individuals had a similar, severe infantile and often lethal multi-systemic disorder that included hypotonia, developmental delay, hypertrophic cardiomyopathy, progressive epileptic encephalopathy, progressive cerebral atrophy, and white matter abnormalities on brain MRI consistent with Leigh syndrome. cDNA studies showed a predominant shortened transcript with skipping of exon 2 and low levels of the normal full-length transcript. Fibroblasts from the affected individuals demonstrated decreased ATP5PO protein, defective assembly of complex V with markedly reduced amounts of peripheral stalk proteins, and complex V hydrolytic activity. Further, expression of human ATP5PO cDNA without exon 2 (hATP5PO-∆ex2) in yeast cells deleted for yATP5 (ATP5PO homolog) was unable to rescue growth on media which requires oxidative phosphorylation when compared to the wild type construct (hATP5PO-WT), indicating that exon 2 deletion leads to a non-functional protein. Collectively, our findings support the pathogenicity of the ATP5PO c.87+3A>G variant, which significantly reduces but does not eliminate complex V activity. These data along with the recent report of an affected individual with ATP5PO variants, add to the evidence that rare biallelic variants in ATP5PO result in defective complex V assembly, function and are associated with Leigh syndrome.

Cbs overdosage is necessary and sufficient to induce cognitive phenotypes in mouse models of Down syndrome and interacts genetically with Dyrk1a.

Identifying dosage-sensitive genes is a key to understand the mechanisms underlying intellectual disability in Down syndrome (DS). The Dp(17Abcg1-Cbs)1Yah DS mouse model (Dp1Yah) shows cognitive phenotypes that need to be investigated to identify the main genetic driver. Here, we report that three copies of the cystathionine-beta-synthase gene (Cbs) in the Dp1Yah mice are necessary to observe a deficit in the novel object recognition (NOR) paradigm. Moreover, the overexpression of Cbs alone is sufficient to induce deficits in the NOR test. Accordingly, overexpressing human CBS specifically in Camk2a-expressing neurons leads to impaired objects discrimination. Altogether, this shows that Cbs overdosage is involved in DS learning and memory phenotypes. To go further, we identified compounds that interfere with the phenotypical consequence of CBS overdosage in yeast. Pharmacological intervention in Tg(CBS) mice with one selected compound restored memory in the NOR test. In addition, using a genetic approach, we demonstrated an epistatic interaction between Cbs and Dyrk1a, another human chromosome 21-located gene (which encodes the dual-specificity tyrosine phosphorylation-regulated kinase 1a) and an already known target for DS therapeutic intervention. Further analysis using proteomic approaches highlighted several molecular pathways, including synaptic transmission, cell projection morphogenesis and actin cytoskeleton, that are affected by DYRK1A and CBS overexpression. Overall, we demonstrated that CBS overdosage underpins the DS-related recognition memory deficit and that both CBS and DYRK1A interact to control accurate memory processes in DS. In addition, our study establishes CBS as an intervention point for treating intellectual deficiencies linked to DS.

Nuclear processing of nascent transcripts determines synthesis of full-length proteins and antigenic peptides.

Peptides presented on major histocompatibility (MHC) class I molecules form an essential part of the immune system's capacity to detect virus-infected or transformed cells. Earlier works have shown that pioneer translation peptides (PTPs) for the MHC class I pathway are as efficiently produced from introns as from exons, or from mRNAs targeted for the nonsense-mediated decay pathway. The production of PTPs is a target for viral immune evasion but the underlying molecular mechanisms that govern this non-canonical translation are unknown. Here, we have used different approaches to show how events taking place on the nascent transcript control the synthesis of PTPs and full-length proteins. By controlling the subcellular interaction between the G-quadruplex structure (G4) of a gly-ala encoding mRNA and nucleolin (NCL) and by interfering with mRNA maturation using multiple approaches, we demonstrate that antigenic peptides derive from a nuclear non-canonical translation event that is independently regulated from the synthesis of full-length proteins. Moreover, we show that G4 are exploited to control mRNA localization and translation by distinguishable mechanisms that are targets for viral immune evasion.

The importance of naturally attenuated SARS-CoV-2in the fight against COVID-19.

The current SARS-CoV-2 pandemic is wreaking havoc throughout the world and has rapidly become a global health emergency. A central question concerning COVID-19 is why some individuals become sick and others not. Many have pointed already at variation in risk factors between individuals. However, the variable outcome of SARS-CoV-2 infections may, at least in part, be due also to differences between the viral subspecies with which individuals are infected. A more pertinent question is how we are to overcome the current pandemic. A vaccine against SARS-CoV-2 would offer significant relief, although vaccine developers have warned that design, testing and production of vaccines may take a year if not longer. Vaccines are based on a handful of different designs (i), but the earliest vaccines were based on the live, attenuated virus. As has been the case for other viruses during earlier pandemics, SARS-CoV-2 will mutate and may naturally attenuate over time (ii). What makes the current pandemic unique is that, thanks to state-of-the-art nucleic acid sequencing technologies, we can follow in detail how SARS-CoV-2 evolves while it spreads. We argue that knowledge of naturally emerging attenuated SARS-CoV-2 variants across the globe should be of key interest in our fight against the pandemic.

An Overview of In Vivo and In Vitro Models for Autosomal Dominant Polycystic Kidney Disease: A Journey from 3D-Cysts to Mini-Pigs.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common inheritable cause of end stage renal disease and, as of today, only a single moderately effective treatment is available for patients. Even though ADPKD research has made huge progress over the last decades, the precise disease mechanisms remain elusive. However, a wide variety of cellular and animal models have been developed to decipher the pathophysiological mechanisms and related pathways underlying the disease. As none of these models perfectly recapitulates the complexity of the human disease, the aim of this review is to give an overview of the main tools currently available to ADPKD researchers, as well as their main advantages and limitations.

In Cellulo Protein-mRNA Interaction Assay to Determine the Action of G-Quadruplex-Binding Molecules.

Protein-RNA interactions (PRIs) control pivotal steps in RNA biogenesis, regulate multiple physiological and pathological cellular networks, and are emerging as important drug targets. However, targeting of specific protein-RNA interactions for therapeutic developments is still poorly advanced. Studies and manipulation of these interactions are technically challenging and in vitro drug screening assays are often hampered due to the complexity of RNA structures. The binding of nucleolin (NCL) to a G-quadruplex (G4) structure in the messenger RNA (mRNA) of the Epstein-Barr virus (EBV)-encoded EBNA1 has emerged as an interesting therapeutic target to interfere with immune evasion of EBV-associated cancers. Using the NCL-EBNA1 mRNA interaction as a model, we describe a quantitative proximity ligation assay (PLA)-based in cellulo approach to determine the structure activity relationship of small chemical G4 ligands. Our results show how different G4 ligands have different effects on NCL binding to G4 of the EBNA1 mRNA and highlight the importance of in-cellulo screening assays for targeting RNA structure-dependent interactions.

Epstein-Barr virus-encoded EBNA1 and ZEBRA: targets for therapeutic strategies against EBV-carrying cancers.

The EBV-encoded EBNA1 was first discovered 40 years ago, approximately 10 years after the presence of EBV had been demonstrated in Burkitt's lymphoma cells. It took another 10 years before the functions of EBNA1 in maintaining the viral genome were revealed, and it has since been shown to be an essential viral factor expressed in all EBV-carrying cells. Apart from serving to maintain the viral episome and to control viral replication and gene expression, EBNA1 also harbours a cis-acting mechanism that allows virus-carrying host cells to evade the immune system. This relates to a particular glycine-alanine repeat (GAr) within EBNA1 that has the capacity to suppress antigen presentation to the major histocompatibility complex (MHC) class I pathway. We discuss the role of the GAr sequence at the level of mRNA translation initiation, rather than at the protein level, as at least part of the mechanism to avoid MHC presentation. Interfering with this mechanism has become the focus of the development of immune-based therapies against EBV-carrying cancers, and some lead compounds that affect translation of GAr-carrying mRNAs have been identified. In addition, we describe the EBV-encoded ZEBRA factor and the switch from the latent to the lytic cycle as an alternative virus-specific target for treating EBV-carrying cancers. Understanding the molecular mechanisms of how EBNA1 and ZEBRA interfere with cellular pathways not only opens new therapeutic approaches but continues to reveal new cell-biological insights on the interplay between host and virus. This review is a tale of discoveries relating to how EBNA1 and ZEBRA have emerged as targets for specific cancer therapies against EBV-carrying diseases, and serves as an illustration of how mRNA translation can play roles in future immune-based strategies to target viral disease.

Using yeast to model calcium-related diseases: example of the Hailey-Hailey disease.

Cross-complementation studies offer the possibility to overcome limitations imposed by the inherent complexity of multicellular organisms in the study of human diseases, by taking advantage of simpler model organisms like the budding yeast Saccharomyces cerevisiae. This review deals with, (1) the use of S. cerevisiae as a model organism to study human diseases, (2) yeast-based screening systems for the detection of disease modifiers, (3) Hailey-Hailey as an example of a calcium-related disease, and (4) the presentation of a yeast-based model to search for chemical modifiers of Hailey-Hailey disease. The preliminary experimental data presented and discussed here show that it is possible to use yeast as a model system for Hailey-Hailey disease and suggest that in all likelihood, yeast has the potential to reveal candidate drugs for the treatment of this disorder. This article is part of a Special Issue entitled: Calcium signaling in health and disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.

The antiprion compound 6-aminophenanthridine inhibits the protein folding activity of the ribosome by direct competition.

Domain V of the 23S/25S/28S rRNA of the large ribosomal subunit constitutes the active center for the protein folding activity of the ribosome (PFAR). Using in vitro transcribed domain V rRNAs from Escherichia coli and Saccharomyces cerevisiae as the folding modulators and human carbonic anhydrase as a model protein, we demonstrate that PFAR is conserved from prokaryotes to eukaryotes. It was shown previously that 6-aminophenanthridine (6AP), an antiprion compound, inhibits PFAR. Here, using UV cross-linking followed by primer extension, we show that the protein substrates and 6AP interact with a common set of nucleotides on domain V of 23S rRNA. Mutations at the interaction sites decreased PFAR and resulted in loss or change of the binding pattern for both the protein substrates and 6AP. Moreover, kinetic analysis of human carbonic anhydrase refolding showed that 6AP decreased the yield of the refolded protein but did not affect the rate of refolding. Thus, we conclude that 6AP competitively occludes the protein substrates from binding to rRNA and thereby inhibits PFAR. Finally, we propose a scheme clarifying the mechanism by which 6AP inhibits PFAR.

A yeast-based assay identifies drugs active against human mitochondrial disorders.

Due to the lack of relevant animal models, development of effective treatments for human mitochondrial diseases has been limited. Here we establish a rapid, yeast-based assay to screen for drugs active against human inherited mitochondrial diseases affecting ATP synthase, in particular NARP (neuropathy, ataxia, and retinitis pigmentosa) syndrome. This method is based on the conservation of mitochondrial function from yeast to human, on the unique ability of yeast to survive without production of ATP by oxidative phosphorylation, and on the amenability of the yeast mitochondrial genome to site-directed mutagenesis. Our method identifies chlorhexidine by screening a chemical library and oleate through a candidate approach. We show that these molecules rescue a number of phenotypes resulting from mutations affecting ATP synthase in yeast. These compounds are also active on human cybrid cells derived from NARP patients. These results validate our method as an effective high-throughput screening approach to identify drugs active in the treatment of human ATP synthase disorders and suggest that this type of method could be applied to other mitochondrial diseases.

[Chemobiology at happy hour: yeast as a model for pharmacological screening]. / Chémobiologie à l'happy hour - La levure comme modèle de criblage pharmacologique.

Since its discovery and description by Louis Pasteur, the budding yeast Saccharomyces cerevisiae, which was used for thousands of years for alcoholic fermentation and as a leavening agent, has become a popular model system in biology. One of the reasons for this popularity is the strong conservation from yeast to human of most of the pathways controlling cell growth and fate. In addition, at least 30 % of human genes involved in diseases have a functional homolog in yeast. Hence, yeast is now widely used for modelling and deciphering physiopathological mechanisms as well as for developing pharmacological approaches like phenotype-based drug screening. Three examples of such yeast-based chemobiological studies are presented.

The hide-and-seek game of the oncogenic Epstein-Barr virus-encoded EBNA1 protein with the immune system: An RNA G-quadruplex tale.

The Epstein-Barr virus (EBV) is the first oncogenic virus described in human. EBV infects more than 90% of the human population worldwide, but most EBV infections are asymptomatic. After the primary infection, the virus persists lifelong in the memory B cells of the infected individuals. Under certain conditions the virus can cause several human cancers, that include lymphoproliferative disorders such as Burkitt and Hodgkin lymphomas and non-lymphoid malignancies such as 100% of nasopharyngeal carcinoma and 10% of gastric cancers. Each year, about 200,000 EBV-related cancers emerge, hence accounting for at least 1% of worldwide cancers. Like all gammaherpesviruses, EBV has evolved a strategy to escape the host immune system. This strategy is mainly based on the tight control of the expression of its Epstein-Barr nuclear antigen-1 (EBNA1) protein, the EBV-encoded genome maintenance protein. Indeed, EBNA1 is essential for viral genome replication and maintenance but, at the same time, is also highly antigenic and T cells raised against EBNA1 exist in infected individuals. For this reason, EBNA1 is considered as the Achilles heel of EBV and the virus has seemingly evolved a strategy that employs the binding of nucleolin, a host cell factor, to RNA G-quadruplex (rG4) within EBNA1 mRNA to limit its expression to the minimal level required for function while minimizing immune recognition. This review recapitulates in a historical way the knowledge accumulated on EBNA1 immune evasion and discusses how this rG4-dependent mechanism can be exploited as an intervention point to unveil EBV-related cancers to the immune system.

The different activities of RNA G-quadruplex structures are controlled by flanking sequences.

The role of G-quadruplex (G4) RNA structures is multifaceted and controversial. Here, we have used as a model the EBV-encoded EBNA1 and the Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded LANA1 mRNAs. We have compared the G4s in these two messages in terms of nucleolin binding, nuclear mRNA retention, and mRNA translation inhibition and their effects on immune evasion. The G4s in the EBNA1 message are clustered in one repeat sequence and the G4 ligand PhenDH2 prevents all G4-associated activities. The RNA G4s in the LANA1 message take part in similar multiple mRNA functions but are spread throughout the message. The different G4 activities depend on flanking coding and non-coding sequences and, interestingly, can be separated individually. Together, the results illustrate the multifunctional, dynamic and context-dependent nature of G4 RNAs and highlight the possibility to develop ligands targeting specific RNA G4 functions. The data also suggest a common multifunctional repertoire of viral G4 RNA activities for immune evasion.

Consequences of the pathogenic T9176C mutation of human mitochondrial DNA on yeast mitochondrial ATP synthase.

Several human neurological disorders have been associated with various mutations affecting mitochondrial enzymes involved in cellular ATP production. One of these mutations, T9176C in the mitochondrial DNA (mtDNA), changes a highly conserved leucine residue into proline at position 217 of the mitochondrially encoded Atp6p (or a) subunit of the F1FO-ATP synthase. The consequences of this mutation on the mitochondrial ATP synthase are still poorly defined. To gain insight into the primary pathogenic mechanisms induced by T9176C, we have investigated the consequences of this mutation on the ATP synthase of yeast where Atp6p is also encoded by the mtDNA. In vitro, yeast atp6-T9176C mitochondria showed a 30% decrease in the rate of ATP synthesis. When forcing the F1FO complex to work in the reverse mode, i.e. F1-catalyzed hydrolysis of ATP coupled to proton transport out of the mitochondrial matrix, the mutant showed a normal proton-pumping activity and this activity was fully sensitive to oligomycin, an inhibitor of the ATP synthase proton channel. However, under conditions of maximal ATP hydrolytic activity, using non-osmotically protected mitochondria, the mutant ATPase activity was less efficiently inhibited by oligomycin (60% inhibition versus 85% for the wild type control). Blue Native Polyacrylamide Gel Electrophoresis analyses revealed that atp6-T9176C yeast accumulated rather good levels of fully assembled ATP synthase complexes. However, a number of sub-complexes (F1, Atp9p-ring, unassembled alpha-F1 subunits) could be detected as well, presumably because of a decreased stability of Atp6p within the ATP synthase. Although the oxidative phosphorylation capacity was reduced in atp6-T9176C yeast, the number of ATP molecules synthesized per electron transferred to oxygen was similar compared with wild type yeast. It can therefore be inferred that the coupling efficiency within the ATP synthase was mostly unaffected and that the T9176C mutation did not increase the proton permeability of the mitochondrial inner membrane.

Anti-prion Drugs Targeting the Protein Folding Activity of the Ribosome Reduce PABPN1 Aggregation.

Prion diseases are caused by the propagation of PrPSc, the pathological conformation of the PrPC prion protein. The molecular mechanisms underlying PrPSc propagation are still unsolved and no therapeutic solution is currently available. We thus sought to identify new anti-prion molecules and found that flunarizine inhibited PrPSc propagation in cell culture and significantly prolonged survival of prion-infected mice. Using an in silico therapeutic repositioning approach based on similarities with flunarizine chemical structure, we tested azelastine, duloxetine, ebastine, loperamide and metixene and showed that they all have an anti-prion activity. Like flunarizine, these marketed drugs reduced PrPSc propagation in cell culture and in mouse cerebellum organotypic slice culture, and inhibited the protein folding activity of the ribosome (PFAR). Strikingly, some of these drugs were also able to alleviate phenotypes due to PABPN1 nuclear aggregation in cell and Drosophila models of oculopharyngeal muscular dystrophy (OPMD). These data emphasize the therapeutic potential of anti-PFAR drugs for neurodegenerative and neuromuscular proteinopathies.

Mitochondrial ATP synthase disorders: molecular mechanisms and the quest for curative therapeutic approaches.

In mammals, the majority of cellular ATP is produced by the mitochondrial F1F(O)-ATP synthase through an elaborate catalytic mechanism. While most subunits of this enzymatic complex are encoded by the nuclear genome, a few essential components are encoded in the mitochondrial genome. The biogenesis of this multi-subunit enzyme is a sophisticated multi-step process that is regulated on levels of transcription, translation and assembly. Defects that result in diminished abundance or functional impairment of the F1F(O)-ATP synthase can cause a variety of severe neuromuscular disorders. Underlying mutations have been identified in both the nuclear and the mitochondrial DNA. The pathogenic mechanisms are only partially understood. Currently, the therapeutic options are extremely limited. Alternative methods of treatment have however been proposed, but still encounter several technical difficulties. The application of novel scientific approaches promises to deepen our understanding of the molecular mechanisms of the ATP synthase, unravel novel therapeutic pathways and improve the unfortunate situation of the patients suffering from such diseases.

Synthesis of conjugates of 6-aminophenanthridine and guanabenz, two structurally unrelated prion inhibitors, for the determination of their cellular targets by affinity chromatography.

The synthesis of affinity matrices for 6-aminophenanthridine (6AP) and 2,6-dichlorobenzylidenaminoguanidine (Guanabenz, GA), two unrelated prion inhibitors, is described. In both cases, the same simple spacer, epsilon-aminocaproylaminopentanol, was introduced by a Mitsunobu reaction and the choice of the anchoring position of the linker was determined by the study of the residual antiprion activity of the corresponding 6AP or GA conjugates. Very recently, these two affinity matrices were used for chromatography assays leading to the identification of ribosome (via the rRNA) as a common target of these two antiprion drugs. Here, we show, using competition experiments with Quinacrine (QC) and Chlorpromazine (CPZ), two other antiprion drugs, that QC, but not CPZ, may also directly target the rRNA.