HEATR3 variants impair nuclear import of uL18 (RPL5) and drive Diamond-Blackfan anemia.

The congenital bone marrow failure syndrome Diamond-Blackfan anemia (DBA) is typically associated with variants in ribosomal protein (RP) genes impairing erythroid cell development. Here we report multiple individuals with biallelic HEATR3 variants exhibiting bone marrow failure, short stature, facial and acromelic dysmorphic features, and intellectual disability. These variants destabilize a protein whose yeast homolog is known to synchronize the nuclear import of RPs uL5 (RPL11) and uL18 (RPL5), which are both critical for producing ribosomal subunits and for stabilizing the p53 tumor suppressor when ribosome biogenesis is compromised. Expression of HEATR3 variants or repression of HEATR3 expression in primary cells, cell lines of various origins, and yeast models impairs growth, differentiation, pre-ribosomal RNA processing, and ribosomal subunit formation reminiscent of DBA models of large subunit RP gene variants. Consistent with a role of HEATR3 in RP import, HEATR3-depleted cells or patient-derived fibroblasts display reduced nuclear accumulation of uL18. Hematopoietic progenitor cells expressing HEATR3 variants or small-hairpin RNAs knocking down HEATR3 synthesis reveal abnormal acceleration of erythrocyte maturation coupled to severe proliferation defects that are independent of p53 activation. Our study uncovers a new pathophysiological mechanism leading to DBA driven by biallelic HEATR3 variants and the destabilization of a nuclear import protein important for ribosome biogenesis.

Specific Circular RNA Signature of Endothelial Cells: Potential Implications in Vascular Pathophysiology.

Circular RNAs (circRNAs) are a recently characterized family of gene transcripts forming a covalently closed loop of single-stranded RNA. The extent of their potential for fine-tuning gene expression is still being discovered. Several studies have implicated certain circular RNAs in pathophysiological processes within vascular endothelial cells and cancer cells independently. However, to date, no comparative study of circular RNA expression in different types of endothelial cells has been performed and analysed through the lens of their central role in vascular physiology and pathology. In this work, we analysed publicly available and original RNA sequencing datasets from arterial, veinous, and lymphatic endothelial cells to identify common and distinct circRNA expression profiles. We identified 4713 distinct circRNAs in the compared endothelial cell types, 95% of which originated from exons. Interestingly, the results show that the expression profile of circular RNAs is much more specific to each cell type than linear RNAs, and therefore appears to be more suitable for distinguishing between them. As a result, we have discovered a specific circRNA signature for each given endothelial cell type. Furthermore, we identified a specific endothelial cell circRNA signature that is composed four circRNAs: circCARD6, circPLXNA2, circCASC15 and circEPHB4. These circular RNAs are produced by genes that are related to endothelial cell migration pathways and cancer progression. More detailed studies of their functions could lead to a better understanding of the mechanisms involved in physiological and pathological (lymph)angiogenesis and might open new ways to tackle tumour spread through the vascular system.

Mitochondrial Ribosomal Protein MRPS15 Is a Component of Cytosolic Ribosomes and Regulates Translation in Stressed Cardiomyocytes.

Regulation of mRNA translation is a crucial step in controlling gene expression in stressed cells, impacting many pathologies, including heart ischemia. In recent years, ribosome heterogeneity has emerged as a key control mechanism driving the translation of subsets of mRNAs. In this study, we investigated variations in ribosome composition in human cardiomyocytes subjected to endoplasmic reticulum stress induced by tunicamycin treatment. Our findings demonstrate that this stress inhibits global translation in cardiomyocytes while activating internal ribosome entry site (IRES)-dependent translation. Analysis of translating ribosome composition in stressed and unstressed cardiomyocytes was conducted using mass spectrometry. We observed no significant changes in ribosomal protein composition, but several mitochondrial ribosomal proteins (MRPs) were identified in cytosolic polysomes, showing drastic variations between stressed and unstressed cells. The most notable increase in polysomes of stressed cells was observed in MRPS15. Its interaction with ribosomal proteins was confirmed by proximity ligation assay (PLA) and immunoprecipitation, suggesting its intrinsic role as a ribosomal component during stress. Knock-down or overexpression experiments of MRPS15 revealed its role as an activator of IRES-dependent translation. Furthermore, polysome profiling after immunoprecipitation with anti-MRPS15 antibody revealed that the "MRPS15 ribosome" is specialized in translating mRNAs involved in the unfolded protein response.

High Level of Staufen1 Expression Confers Longer Recurrence Free Survival to Non-Small Cell Lung Cancer Patients by Promoting THBS1 mRNA Degradation.

Stau1 is a pluripotent RNA-binding protein that is responsible for the post-transcriptional regulation of a multitude of transcripts. Here, we observed that lung cancer patients with a high Stau1 expression have a longer recurrence free survival. Strikingly, Stau1 did not impair cell proliferation in vitro, but rather cell migration and cell adhesion. In vivo, Stau1 depletion favored tumor progression and metastases development. In addition, Stau1 depletion strongly impaired vessel maturation. Among a panel of candidate genes, we specifically identified the mRNA encoding the cell adhesion molecule Thrombospondin 1 (THBS1) as a new target for Staufen-mediated mRNA decay. Altogether, our results suggest that regulation of THBS1 expression by Stau1 may be a key process involved in lung cancer progression.

Circular RNA, the Key for Translation.

It was thought until the 1990s that the eukaryotic translation machinery was unable to translate a circular RNA. However internal ribosome entry sites (IRESs) and m6A-induced ribosome engagement sites (MIRESs) were discovered, promoting 5' end-independent translation initiation. Today a new family of so-called "noncoding" circular RNAs (circRNAs) has emerged, revealing the pivotal role of 5' end-independent translation. CircRNAs have a strong impact on translational control via their sponge function, and form a new mRNA family as they are translated into proteins with pathophysiological roles. While there is no more doubt about translation of covalently closed circRNA, the linearity of canonical mRNA is only theoretical: it has been shown for more than thirty years that polysomes exhibit a circular form and mRNA functional circularization has been demonstrated in the 1990s by the interaction of initiation factor eIF4G with poly(A) binding protein. More recently, additional mechanisms of 3'-5' interaction have been reported, including m6A modification. Functional circularization enhances translation via ribosome recycling and acceleration of the translation initiation rate. This update of covalently and noncovalently closed circular mRNA translation landscape shows that RNA with circular shape might be the rule for translation with an important impact on disease development and biotechnological applications.

Lymphatic Vasculature Requires Estrogen Receptor-α Signaling to Protect From Lymphedema.

OBJECTIVE: Estrogens exert beneficial effect on the blood vascular system. However, their role on the lymphatic system has been poorly investigated. We studied the protective effect of the 17ß estradiol-the most potent endogenous estrogen-in lymphedema-a lymphatic dysfunction, which results in a massive fluid and fat accumulation in the limb. APPROACH AND RESULTS: Screening of DNA motifs able to mobilize ERs (estrogen receptors) and quantitative real-time polymerase chain reaction analysis revealed that estradiol promotes transcriptional activation of lymphangiogenesis-related gene expression including VEGF (vascular endothelial growth factor)-D, VEGFR (VEGF receptor)-3, lyve-1, and HASs (hyaluronan synthases). Using an original model of secondary lymphedema, we observed a protective effect of estradiol on lymphedema by reducing dermal backflow-a representative feature of the pathology. Blocking ERα by tamoxifen-the selective estrogen modulator-led to a remodeling of the lymphatic network associated with a strong lymphatic leakage. Moreover, the protection of lymphedema by estradiol treatment was abrogated by the endothelial deletion of the receptor ERα in Tie2-Cre; ERαlox/lox mice, which exhibit dilated lymphatic vessels. This remodeling correlated with a decrease in lymphangiogenic gene expression. In vitro, blocking ERα by tamoxifen in lymphatic endothelial cells decreased cell-cell junctions, inhibited migration and sprouting, and resulted in an inhibition of Erk but not of Akt phosphorylation. CONCLUSIONS: Estradiol protection from developing lymphedema is mediated by an activation of its receptor ERα and is antagonized by tamoxifen. These findings reveal a new facet of the estrogen influence in the management of the lymphatic system and provide more evidence that secondary lymphedema is worsened by hormone therapy.

IRES Trans-Acting Factors, Key Actors of the Stress Response.

The cellular stress response corresponds to the molecular changes that a cell undergoes in response to various environmental stimuli. It induces drastic changes in the regulation of gene expression at transcriptional and posttranscriptional levels. Actually, translation is strongly affected with a blockade of the classical cap-dependent mechanism, whereas alternative mechanisms are activated to support the translation of specific mRNAs. A major mechanism involved in stress-activated translation is the internal ribosome entry site (IRES)-driven initiation. IRESs, first discovered in viral mRNAs, are present in cellular mRNAs coding for master regulators of cell responses, whose expression must be tightly controlled. IRESs allow the translation of these mRNAs in response to different stresses, including DNA damage, amino-acid starvation, hypoxia or endoplasmic reticulum stress, as well as to physiological stimuli such as cell differentiation or synapse network formation. Most IRESs are regulated by IRES trans-acting factor (ITAFs), exerting their action by at least nine different mechanisms. This review presents the history of viral and cellular IRES discovery as well as an update of the reported ITAFs regulating cellular mRNA translation and of their different mechanisms of action. The impact of ITAFs on the coordinated expression of mRNA families and consequences in cell physiology and diseases are also highlighted.

Hypoxia and ER stress promote Staufen1 expression through an alternative translation mechanism.

Under physiological stress conditions the cell protects itself through a global blockade on cap-dependent translation of mRNA. This allows cap-independent mechanisms such as internal ribosome entry site (IRES)-mediated translation to take over and initiate the translation of a specific pool of mRNAs that encode proteins involved in protecting the cell from stress. Staufen 1 (Stau1) is an RNA-binding protein that has been previously implicated in the regulation of stress granule formation and therefore could play a key role in protecting the cell against stress stimuli such as oxidative and endoplasmic reticulum (ER) stress. We hypothesized that Stau1 mRNA could, like many stress response genes, contain an IRES in its 5'UTR. Here we describe that a bona fide IRES element is present in the 5'UTR of Stau1 mRNA, which is activated under hypoxic and ER stress conditions. Further, we show that the activity of PERK kinase, a major effector of the ER stress response, is required for Stau1 IRES-mediated translation during ER stress. These results suggest that Stau1 is a stress response gene that remains efficiently translated during hypoxia and ER stress despite the substantial global inhibition of cap-dependent protein translation, promoting cell recovery following stress.

VEGF-A mRNA processing, stability and translation: a paradigm for intricate regulation of gene expression at the post-transcriptional level.

Vascular Endothelial Growth Factor A (VEGF-A) is a potent secreted mitogen crucial for physiological and pathological angiogenesis. Post-transcriptional regulation of VEGF-A occurs at multiple levels. Firstly, alternative splicing gives rise to different transcript variants encoding diverse isoforms that exhibit distinct biological properties with regard to receptor binding and extra-cellular localization. Secondly, VEGF-A mRNA stability is regulated by effectors such as hypoxia or growth factors through the binding of stabilizing and destabilizing proteins at AU-rich elements located in the 3'-untranslated region. Thirdly, translation of VEGF-A mRNA is a controlled process involving alternative initiation codons, internal ribosome entry sites (IRESs), an upstream open reading frame (uORF), miRNA targeting and a riboswitch in the 3' untranslated region. These different levels of regulation cooperate for the crucial fine-tuning of the expression of VEGF-A variants. This review will be focused on our current knowledge of the complex post-transcriptional regulatory switches that modulate the cellular VEGF-A level, a paradigmatic model of post-transcriptional regulation.

Translational control by long non-coding RNAs.

Long non-coding (lnc) RNAs, once considered as junk and useless, are now broadly recognized to have major functions in the cell. LncRNAs are defined as non-coding RNAs of more than 200 nucleotides, regulate all steps of gene expression. Their origin is diverse, they can arise from intronic, intergenic or overlapping region, in sense or antisense direction. LncRNAs are mainly described for their action on transcription, while their action at the translational level is more rarely cited. However, the bibliography in the field is more and more abundant. The present synopsis of lncRNAs involved in the control of translation reveals a wide field of regulation of gene expression, with at least nine distinct molecular mechanisms. Furthermore, it appears that all these lncRNAs are involved in various pathologies including cancer, cardiovascular and neurodegenerative diseases.

Apelin-VEGF-C mRNA delivery as therapeutic for the treatment of secondary lymphedema.

Secondary lymphedema (LD) corresponds to a severe lymphatic dysfunction leading to the accumulation of fluid and fibrotic adipose tissue in a limb. Here, we identified apelin (APLN) as a powerful molecule for regenerating lymphatic function in LD. We identified the loss of APLN expression in the lymphedematous arm compared to the normal arm in patients. The role of APLN in LD was confirmed in APLN knockout mice, in which LD is increased and associated with fibrosis and dermal backflow. This was reversed by intradermal injection of APLN-lentivectors. Mechanistically, APLN stimulates lymphatic endothelial cell gene expression and induces the binding of E2F8 transcription factor to the promoter of CCBE1 that controls VEGF-C processing. In addition, APLN induces Akt and eNOS pathways to stimulate lymphatic collector pumping. Our results show that APLN represents a novel partner for VEGF-C to restore lymphatic function in both initial and collecting vessels. As LD appears after cancer treatment, we validated the APLN-VEGF-C combination using a novel class of nonintegrative RNA delivery LentiFlash® vector that will be evaluated for phase I/IIa clinical trial.

Functional divergence between 2 chemokines is conferred by single amino acid change.

CXCL4 and CXCL4L1 are 2 closely related CXC chemokines that exhibit potent antiangiogenic activity. Because interactions with glycosaminoglycans play a crucial role in chemokines activity, we determined the binding parameters of CXCL4 and CXCL4L1 for heparin, heparan sulfate, and chondroitin sulfate B. We further demonstrated that the Leu67/His67 substitution is critical for the decrease in glycan binding of CXCL4L1 but also for the increase of its angiostatic activities. Using a set of mutants, we show that glycan affinity and angiostatic properties are not completely related. These data are reinforced using a monoclonal antibody that specifically recognizes structural modifications in CXCL4L1 due to the presence of His67 and that blocks its biologic activity. In vivo, half-life and diffusibility of CXCL4L1 compared with CXCL4 is strongly increased. As opposed to CXCL4L1, CXCL4 is preferentially retained at its site of expression. These findings establish that, despite small differences in the primary structure, CXCL4L1 is highly distinct from CXCL4. These observations are not only of great significance for the antiangiogenic activity of CXCL4L1 and for its potential use in clinical development but also for other biologic processes such as inflammation, thrombosis or tissue repair.

Long non-coding RNA Neat1 and paraspeckle components are translational regulators in hypoxia.

Internal ribosome entry sites (IRESs) drive translation initiation during stress. In response to hypoxia, (lymph)angiogenic factors responsible for tissue revascularization in ischemic diseases are induced by the IRES-dependent mechanism. Here, we searched for IRES trans-acting factors (ITAFs) active in early hypoxia in mouse cardiomyocytes. Using knock-down and proteomics approaches, we show a link between a stressed-induced nuclear body, the paraspeckle, and IRES-dependent translation. Furthermore, smiFISH experiments demonstrate the recruitment of IRES-containing mRNA into paraspeckle during hypoxia. Our data reveal that the long non-coding RNA Neat1, an essential paraspeckle component, is a key translational regulator, active on IRESs of (lymph)angiogenic and cardioprotective factor mRNAs. In addition, paraspeckle proteins p54nrb and PSPC1 as well as nucleolin and RPS2, two p54nrb-interacting proteins identified by mass spectrometry, are ITAFs for IRES subgroups. Paraspeckle thus appears as a platform to recruit IRES-containing mRNAs and possibly host IRESome assembly. Polysome PCR array shows that Neat1 isoforms regulate IRES-dependent translation and, more widely, translation of mRNAs involved in stress response.

Pivotal role of translokin/CEP57 in the unconventional secretion versus nuclear translocation of FGF2.

Intracellular trafficking of fibroblast growth factor 2 (FGF2) exhibits two unusual features: (i) it is secreted despite the lack of signal peptide and (ii) it can translocate to the nucleus after interaction with high- and low-affinity receptors on the cell surface, although it does not possess any classical nuclear localization signal. This nuclear translocation constitutes an important part of the response to the growth factor. Previously, we identified Translokin/CEP57, an FGF2 binding partner, as an intracellular mediator of FGF2 trafficking, which is essential for the nuclear translocation of the growth factor. Here, we report the identification of four Translokin partners: sorting nexin 6, Ran-binding protein M and the kinesins KIF3A and KIF3B. These proteins, through their interaction with Translokin, are involved in two exclusive complexes allowing the bidirectional trafficking of FGF2. Thus, Translokin plays a pivotal role in this original mechanism. In addition, we show that FGF2 secretion is regulated by a negative loop, retro-controlled by FGF receptor and involving FGF2 itself.

The VEGF IRESes are differentially susceptible to translation inhibition by miR-16.

Experiments with EMCV (Encephalomyocarditis virus) internal ribosome entry sites (IRESes) have shown that microRNAs (miRs) are unable to inhibit IRES driven translation. However, it is accepted that miRs can inhibit translation through multiple mechanisms, only some of which require interaction with the 5' cap structure. In this report, we first validate the targeting of miR-16 to a predicted binding site in the VEGF 3'UTR. We developed a series of experiments to ascertain whether or not miR-16 can inhibit translation of transcripts driven by either of the VEGF IRESes. Our results indicate that cellular IRESes can be classified as both sensitive and insensitive to miR control. While VEGF IRES-A activity was not altered by miR-16 targeting to the 3'UTR, IRES-B was susceptible to miR-16 inhibition. Taken together with previous results that show that IRES-B selectively translates the CUG initiated VEGF-121 isoform, we can conclude that the existence of two differentially susceptible IRESes in the VEGF 5'UTR leads to even more complex regulatory control of VEGF isoform production. This study demonstrates for the first time the inhibition of cellular IRES driven translation by a miR.

Alternative-splicing-based bicistronic vectors for ratio-controlled protein expression and application to recombinant antibody production.

In the last decade polycistronic vectors have become essential tools for both basic science and gene therapy applications. In order to co-express heterologous polypeptides, different systems have been developed from Internal Ribosome Entry Site (IRES) based vectors to the use of the 2A peptide. Unfortunately, these methods are not fully suitable for the efficient and reproducible modulation of the ratio between the proteins of interest. Here we describe a novel bicistronic vector type based on the use of alternative splicing. By modifying the consensus sequence that governs splicing, we demonstrate that the ratio between the synthesized proteins could easily vary from 1 : 10 to 10 : 1. We have established this system with luciferase genes and we extended its application to the production of recombinant monoclonal antibodies. We have shown that these vectors could be used in several typical cell lines with similar efficiencies. We also present an adaptation of these vectors to hybrid alternative splicing/IRES constructs that allow a ratio-controlled expression of proteins of interest in stably transfected cell lines.

Coordinating Effect of VEGFC and Oleic Acid Participates to Tumor Lymphangiogenesis.

In cancer, the lymphatic system is hijacked by tumor cells that escape from primary tumor and metastasize to the sentinel lymph nodes. Tumor lymphangiogenesis is stimulated by the vascular endothelial growth factors-C (VEGFC) after binding to its receptor VEGFR-3. However, how VEGFC cooperates with other molecules to promote lymphatics growth has not been fully determined. We showed that lymphangiogenesis developed in tumoral lesions and in surrounding adipose tissue (AT). Interestingly, lymphatic vessel density correlated with an increase in circulating free fatty acids (FFA) in the lymph from tumor-bearing mice. We showed that adipocyte-released FFA are uploaded by lymphatic endothelial cells (LEC) to stimulate their sprouting. Lipidomic analysis identified the monounsaturated oleic acid (OA) as the major circulating FFA in the lymph in a tumoral context. OA transporters FATP-3, -6 and CD36 were only upregulated on LEC in the presence of VEGFC showing a collaborative effect of these molecules. OA stimulates fatty acid ß-oxidation in LECs, leading to increased AT lymphangiogenesis. Our results provide new insights on the dialogue between tumors and adipocytes via the lymphatic system and identify a key role for adipocyte-derived FFA in the promotion of lymphangiogenesis, revealing novel therapeutic opportunities for inhibitors of lymphangiogenesis in cancer.

Sex Hormones in Lymphedema.

Lymphedema is a disorder of the lymphatic vascular system characterized by impaired lymphatic return resulting in swelling of the extremities and accumulation of undrained interstitial fluid/lymph that results in fibrosis and adipose tissue deposition in the limb. Whereas it is clearly established that primary lymphedema is sex-linked with an average ratio of one male for three females, the role of female hormones, in particular estrogens, has been poorly explored. In addition, secondary lymphedema in Western countries affects mainly women who developed the pathology after breast cancer and undergo through hormone therapy up to five years after cancer surgery. Although lymphadenectomy is identified as a trigger factor, the effect of co-morbidities associated to lymphedema remains elusive, in particular, estrogen receptor antagonists or aromatase inhibitors. In addition, the role of sex hormones and gender has been poorly investigated in the etiology of the pathology. Therefore, this review aims to recapitulate the effect of sex hormones on the physiology of the lymphatic system and to investigate whetherhormone therapy could promote a lymphatic dysfunction leading to lymphedema.

An upstream open reading frame within an IRES controls expression of a specific VEGF-A isoform.

Vascular endothelial growth factor A (VEGF-A) is a potent secreted mitogen critical for physiological and pathological angiogenesis. Regulation of VEGF-A occurs at multiple levels, including transcription, mRNA stabilization, splicing, translation and differential cellular localization of various isoforms. Recent advances in our understanding of the posttranscriptional regulation of VEGF-A are comprised of the identification of stabilizing mRNA-binding proteins and the discovery of two internal ribosomal entry sites (IRES) as well as two alternative initiation codons in the 5'UTR of the VEGF-A mRNA. We have previously reported that VEGF-A translation initiation at both the AUG and CUG codons is dependent on the exon content of the coding region. In this report, we show that the expression of different VEGF-A isoforms is regulated by a small upstream open reading frame (uORF) located within an internal ribosome entry site, which is translated through a cap-independent mechanism. This uORF acts as a cis-regulatory element that regulates negatively the expression of the VEGF 121 isoform. Our data provide a framework for understanding how VEGF-A mRNAs are translated, and how the production of the VEGF 121 isoform is secured under non-hypoxic environmental conditions.

[Do circular RNAs play tricks on us?] / L'ARN circulaire nous joue-t-il des tours ?

RNA has not said its last word with the rise of a new RNA family, circular RNAs (circRNAs). Discovered 25 years ago, circRNAs were initially considered as splicing byproducts. Today it appears that 14% of human genes produce circRNAs, whereas more than 100 000 different circRNAs are expressed. They are produced from coding genes through an alternative splicing mechanism called backsplicing, where an acceptor site is linked with a donor site located downstream. Nuclear circRNAs regulate transcription and splicing of their linear isoform. Cytoplasmic circRNAs, which are predominant, either sequester miRNAs or RNA binding proteins, or are translated via internal initiation mechanisms. CircRNAs may constitute a powerful biotechnogical tool for protein synthesis, as their translation is stable over time. In addition, exogenous circRNAs generate less immune response than their linear counterparts. We will also discuss in this review their biotechnological potential and their roles in pathological processes.

TITLE: L'ARN circulaire nous joue-t-il des tours ? ABSTRACT: L'ARN n'a pas dit son dernier mot… avec l'émergence des ARN circulaires (circARN). Quatorze pour cent des gènes humains produisent en effet des circARN par un mécanisme d'épissage alternatif : le rétro-épissage. Chez l'homme, plus de 100 000 circARN différents ont ainsi été répertoriés. Dans le noyau, ils régulent la transcription ou l'épissage des ARNm, alors que, dans le cytoplasme, ils séquestrent des miARN et des protéines, ou sont traduits par un mécanisme d'initiation interne de la traduction. Ces circARN constituent en fait un outil biotechnologique performant car leur traduction est très stable dans le temps, et les circARN exogènes induisent moins de réponses immunitaires que les ARNm linéaires. Dans cette revue, nous discuterons, après les avoir décrits, du rôle des circARN dans différents processus pathologiques et de leur utilisation en biotechnologie.