The core spliceosomal factor U2AF1 controls cell-fate determination via the modulation of transcriptional networks.

Alternative splicing (AS) plays a central role during cell-fate determination. However, how the core spliceosomal factors (CSFs) are involved in this process is poorly understood. Here, we report the down-regulation of the U2AF1 CSF during stem cell differentiation. To investigate its function in stemness and differentiation, we downregulated U2AF1 in human induced pluripotent stem cells (hiPSCs), using an inducible-shRNA system, to the level found in differentiated ectodermal, mesodermal and endodermal cells. RNA sequencing and computational analysis reveal that U2AF1 down-regulation modulates the expression of development-regulating genes and regulates transcriptional networks involved in cell-fate determination. Furthermore, U2AF1 down-regulation induces a switch in the AS of transcription factors (TFs) required to establish specific cell lineages, and favours the splicing of a differentiated cell-specific isoform of DNMT3B. Our results showed that the differential expression of the core spliceosomal factor U2AF1, between stem cells and the precursors of the three germ layers regulates a cell-type-specific alternative splicing programme and a transcriptional network involved in cell-fate determination via the modulation of gene expression and alternative splicing of transcription regulators.

Rejuvenating senescent and centenarian human cells by reprogramming through the pluripotent state.

Direct reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) provides a unique opportunity to derive patient-specific stem cells with potential applications in tissue replacement therapies and without the ethical concerns of human embryonic stem cells (hESCs). However, cellular senescence, which contributes to aging and restricted longevity, has been described as a barrier to the derivation of iPSCs. Here we demonstrate, using an optimized protocol, that cellular senescence is not a limit to reprogramming and that age-related cellular physiology is reversible. Thus, we show that our iPSCs generated from senescent and centenarian cells have reset telomere size, gene expression profiles, oxidative stress, and mitochondrial metabolism, and are indistinguishable from hESCs. Finally, we show that senescent and centenarian-derived pluripotent stem cells are able to redifferentiate into fully rejuvenated cells. These results provide new insights into iPSC technology and pave the way for regenerative medicine for aged patients.

Antagonistic functions of LMNA isoforms in energy expenditure and lifespan.

Alternative RNA processing of LMNA pre-mRNA produces three main protein isoforms, that is, lamin A, progerin, and lamin C. De novo mutations that favor the expression of progerin over lamin A lead to Hutchinson-Gilford progeria syndrome (HGPS), providing support for the involvement of LMNA processing in pathological aging. Lamin C expression is mutually exclusive with the splicing of lamin A and progerin isoforms and occurs by alternative polyadenylation. Here, we investigate the function of lamin C in aging and metabolism using mice that express only this isoform. Intriguingly, these mice live longer, have decreased energy metabolism, increased weight gain, and reduced respiration. In contrast, progerin-expressing mice show increased energy metabolism and are lipodystrophic. Increased mitochondrial biogenesis is found in adipose tissue from HGPS-like mice, whereas lamin C-only mice have fewer mitochondria. Consistently, transcriptome analyses of adipose tissues from HGPS and lamin C-only mice reveal inversely correlated expression of key regulators of energy expenditure, including Pgc1a and Sfrp5. Our results demonstrate that LMNA encodes functionally distinct isoforms that have opposing effects on energy metabolism and lifespan in mammals.

Long lasting control of viral rebound with a new drug ABX464 targeting Rev - mediated viral RNA biogenesis.

BACKGROUND: Current therapies have succeeded in controlling AIDS pandemic. However, there is a continuing need for new drugs, in particular those acting through new and as yet unexplored mechanisms of action to achieve HIV infection cure. We took advantage of the unique feature of proviral genome to require both activation and inhibition of splicing of viral transcripts to develop molecules capable of achieving long lasting effect on viral replication in humanized mouse models through inhibition of Rev-mediated viral RNA biogenesis. RESULTS: Current HIV therapies reduce viral load during treatment but titers rebound after treatment is discontinued. We devised a new drug that has a long lasting effect after viral load reduction. We demonstrate here that ABX464 compromises HIV replication of clinical isolates of different subtypes without selecting for drug resistance in PBMCs or macrophages. ABX464 alone, also efficiently compromised viral proliferation in two humanized mouse models infected with HIV that require a combination of 3TC, Raltegravir and Tenofovir (HAART) to achieve viral inhibition in current protocols. Crucially, while viral load increased dramatically just one week after stopping HAART treatment, only slight rebound was observed following treatment cessation with ABX464 and the magnitude of the rebound was maintained below to that of HAART for two months after stopping the treatment. Using a system to visualize single HIV RNA molecules in living cells, we show that ABX464 inhibits viral replication by preventing Rev-mediated export of unspliced HIV-1 transcripts to the cytoplasm and by interacting with the Cap Binding Complex (CBC). Deep sequencing of viral RNA from treated cells established that retained viral RNA is massively spliced but importantly, normal cellular splicing is unaffected by the drug. Consistently ABX464 is non-toxic in humans and therefore represents a promising complement to current HIV therapies. CONCLUSIONS: ABX464 represents a novel class of anti-HIV molecules with unique properties. ABX464 has a long lasting effect in humanized mice and neutralizes the expression of HIV-1 proviral genome of infected immune cells including reservoirs and it is therefore a promising drug toward a functional cure of HIV.

ABX464 (Obefazimod) Upregulates miR-124 to Reduce Proinflammatory Markers in Inflammatory Bowel Diseases.

Advanced therapies have transformed the treatment of inflammatory bowel disease; however, many patients fail to respond, highlighting the need for therapies tailored to the underlying cell and molecular disease drivers. The first-in-class oral molecule ABX464 (obefazimod), which selectively upregulates miR-124, has demonstrated its ability to be a well-tolerated treatment with rapid and sustained efficacy in patients with ulcerative colitis (UC). Here, we provide evidence that ABX464 affects the immune system in vitro , in the murine model of inflammatory bowel disease, and in patients with UC. In vitro , ABX464 treatment upregulated miR-124 and led to decreases in proinflammatory cytokines including interleukin (IL) 17 and IL6, and in the chemokine CCL2. Consistently, miR-124 expression was upregulated in the rectal biopsies and blood samples of patients with UC, and a parallel reduction in Th17 cells and IL17a levels was observed in serum samples. In a mouse model of induced intestinal inflammation with dextran sulfate sodium, ABX464 reversed the increases in multiple proinflammatory cytokines in the colon and the upregulation of IL17a secretion in the mesenteric lymph nodes. By upregulating miR-124, ABX464 acts as "a physiological brake" of inflammation, which may explain the efficacy of ABX464 with a favorable tolerability and safety profile in patients with UC.

Replication origins are already licensed in G1 arrested unfertilized sea urchin eggs.

Fertilization relieves the oocyte from a cell cycle arrest, inducing progression towards mitotic cycles. While the signalling pathways involved in oocyte to embryo transition have been widely investigated, how they specifically trigger DNA replication is still unclear. We used sea urchin eggs whose oocytes are arrested in G1 to investigate in vivo the molecular mechanisms regulating initiation of replication after fertilization. Unexpectedly, we found that CDC6, Cdt1 and MCM3, components of the pre-replication complexes (pre-RC) which license origins for replication, were already loaded on female chromatin before fertilization. This is the first demonstration of a cell cycle arrest in metazoan in which chromatin is already licensed for replication. In contrast pre-RC assemble on chromatin post-fertilization as in other organisms. These differences in the timing of pre-RC assembly are accompanied by differences in Cdk2 requirement for DNA replication initiation between female and male chromatin post-fertilization. Finally, we demonstrated that a concomitant inhibition of MAP kinase and ATM/ATR pathways releases the block to DNA synthesis. Our findings provide new insight into the mechanisms contributing to the release of G1 arrest and the control of S-phase entry at fertilization.

Both anti-inflammatory and antiviral properties of novel drug candidate ABX464 are mediated by modulation of RNA splicing.

ABX464 is a first-in-class, clinical-stage, small molecule for oral administration that has shown strong anti-inflammatory effects in the DSS-model for inflammatory bowel disease (IBD) and also prevents replication of the HIV virus. ABX464 which binds to cap binding complex (CBC) has demonstrated safety and efficacy in a phase 2a proof-of-concept clinical trial in patients with Ulcerative colitis. Previously, with limited technologies, it was not possible to quantify the effect of ABX464 on viral and cellular RNA biogenesis. Here, using RNA CaptureSeq and deep sequencing, we report that ABX464 enhances the splicing of HIV RNA in infected PBMCs from six healthy individuals and also the expression and splicing of a single long noncoding RNA to generate the anti-inflammatory miR-124 both ex vivo and in HIV patients. While ABX464 has no effect on pre-mRNA splicing of cellular genes, depletion of CBC complex by RNAi leads to accumulation of intron retention transcripts. These results imply that ABX464 did not inhibit the function of CBC in splicing but rather strengthens it under pathological condition like inflammation and HIV infection. The specific dual ability of ABX464 to generate both anti-inflammatory miR-124 and spliced viral RNA may have applicability for the treatment of both inflammatory diseases and HIV infection.

Cyclin B synthesis and rapamycin-sensitive regulation of protein synthesis during starfish oocyte meiotic divisions.

Translation of cyclin mRNAs represents an important event for proper meiotic maturation and post-fertilization mitoses in many species. Translational control of cyclin B mRNA has been described to be achieved through two separate but related mechanisms: translational repression and polyadenylation. In this paper, we evaluated the contribution of global translational regulation by the cap-dependent translation repressor 4E-BP (eukaryotic initiation factor 4E-binding protein) on the cyclin B protein synthesis during meiotic maturation of the starfish oocytes. We used the immunosupressant drug rapamycin, a strong inhibitor of cap-dependent translation, to check for the involvement of this protein synthesis during this physiological process. Rapamycin was found to prevent dissociation of 4E-BP from the initiation factor eIF4E and to suppress correlatively a burst of global protein synthesis occurring at the G2/M transition. The drug had no effect on first meiotic division but defects in meiotic spindle formation prevented second polar body emission, demonstrating that a rapamycin-sensitive pathway is involved in this mechanism. While rapamycin affected the global protein synthesis, the drug altered neither the specific translation of cyclin B mRNA nor the expression of the Mos protein. The expression of these two proteins was correlated with the phosphorylation and the dissociation of the cytoplasmic polyadenylation element-binding protein from eIF4E.

SIRT3, a mitochondrial NAD⁺-dependent deacetylase, is involved in the regulation of myoblast differentiation.

Sirtuin 3 (SIRT3), one of the seven mammalian sirtuins, is a mitochondrial NAD+-dependent deacetylase known to control key metabolic pathways. SIRT3 deacetylases and activates a large number of mitochondrial enzymes involved in the respiratory chain, in ATP production, and in both the citric acid and urea cycles. We have previously shown that the regulation of myoblast differentiation is tightly linked to mitochondrial activity. Since SIRT3 modulates mitochondrial activity, we decide to address its role during myoblast differentiation. For this purpose, we first investigated the expression of endogenous SIRT3 during C2C12 myoblast differentiation. We further studied the impact of SIRT3 silencing on both the myogenic potential and the mitochondrial activity of C2C12 cells. We showed that SIRT3 protein expression peaked at the onset of myoblast differentiation. The inhibition of SIRT3 expression mediated by the stable integration of SIRT3 short inhibitory RNA (SIRT3shRNA) in C2C12 myoblasts, resulted in: 1) abrogation of terminal differentiation - as evidenced by a marked decrease in the myoblast fusion index and a significant reduction of Myogenin, MyoD, Sirtuin 1 and Troponin T protein expression - restored upon MyoD overexpression; 2) a decrease in peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and citrate synthase protein expression reflecting an alteration of mitochondrial density; and 3) an increased production of reactive oxygen species (ROS) mirrored by the decreased activity of manganese superoxide dismutase (MnSOD). Altogether our data demonstrate that SIRT3 mainly regulates myoblast differentiation via its influence on mitochondrial activity.

MBNL1 and RBFOX2 cooperate to establish a splicing programme involved in pluripotent stem cell differentiation.

Reprogramming somatic cells into induced pluripotent stem cells (iPSCs) has provided huge insight into the pathways, mechanisms and transcription factors that control differentiation. Here we use high-throughput RT-PCR technology to take a snapshot of splicing changes in the full spectrum of high- and low-expressed genes during induction of fibroblasts, from several donors, into iPSCs and their subsequent redifferentiation. We uncover a programme of concerted alternative splicing changes involved in late mesoderm differentiation and controlled by key splicing regulators MBNL1 and RBFOX2. These critical splicing adjustments arise early in vertebrate evolution and remain fixed in at least 10 genes (including PLOD2, CLSTN1, ATP2A1, PALM, ITGA6, KIF13A, FMNL3, PPIP5K1, MARK2 and FNIP1), implying that vertebrates require alternative splicing to fully implement the instructions of transcriptional control networks.

Unraveling cell type-specific and reprogrammable human replication origin signatures associated with G-quadruplex consensus motifs.

DNA replication is highly regulated, ensuring faithful inheritance of genetic information through each cell cycle. In metazoans, this process is initiated at many thousands of DNA replication origins whose cell type-specific distribution and usage are poorly understood. We exhaustively mapped the genome-wide location of replication origins in human cells using deep sequencing of short nascent strands and identified ten times more origin positions than we expected; most of these positions were conserved in four different human cell lines. Furthermore, we identified a consensus G-quadruplex-forming DNA motif that can predict the position of DNA replication origins in human cells, accounting for their distribution, usage efficiency and timing. Finally, we discovered a cell type-specific reprogrammable signature of cell identity that was revealed by specific efficiencies of conserved origin positions and not by the selection of cell type-specific subsets of origins.

Nuclear envelope breakdown may deliver an inhibitor of protein phosphatase 1 which triggers cyclin B translation in starfish oocytes.

In vertebrates, enhanced translation of mRNAs in oocytes and early embryos entering M-phase is thought to occur through polyadenylation, involving binding, hyperphosphorylation and proteolytic degradation of Aurora-activated CPEB. In starfish, an unknown component of the oocyte nucleus is required for cyclin B synthesis following the release of G2/prophase block by hormonal stimulation. We have found that CPEB cannot be hyperphosphorylated following hormonal stimulation in starfish oocytes from which the nucleus has been removed. Activation of Aurora kinase, known to interact with protein phosphatase 1 and its specific inhibitor Inh-2, is also prevented. The microinjection of Inh-2 restores Aurora activation, CPEB hyperphosphorylation and cyclin B translation in enucleated oocytes. Nevertheless, we provide evidence that CPEB is in fact hyperphosphorylated by cdc2, without apparent involvement of Aurora or MAP kinase, and that cyclin B synthesis can be stimulated without previous degradation of phosphorylated CPEB. Thus, the regulation of cyclin B synthesis necessary for progression through meiosis can be explained by an equilibrium between CPEB phosphorylation and dephosphorylation, and both aspects of this control may rely on the sole activation of Cdc2 and subsequent nuclear breakdown.

Regulation of Cdc42-mediated morphological effects: a novel function for p53.

The tumour suppressor functions of p53 that are important for its activity depend on its role as a cell cycle arrest mediator and apoptosis inducer. Here we identify a novel function for p53 in regulating cell morphology and movement. We investigated the overall effect of p53 on morphological changes induced by RhoA, Rac1 and Cdc42 GTPases in mouse embryonic fibroblasts (MEFs). Interestingly, p53 exerted a selective effect on Cdc42-mediated cell functions. (i) Both overexpression of wild-type p53 and activation of endogenous p53 counteracted Cdc42-induced filopodia formation. Conversely, p53-deficient MEFs exhibited constitutive membrane filopodia. Mechanistic studies indicate that p53 prevents the initiating steps of filopodia formation downstream of Cdc42. (ii) Over expression of p53 modulates cell spreading of MEFs on fibronectin. (iii) During cell migration, the reorientation of the Golgi apparatus in the direction of movement is abolished by wild-type p53 expression, thus preventing cell polarity. Our data demonstrate a previously uncharacterized role for p53 in regulating Cdc42-dependent cell effects that control actin cytoskeletal dynamics and cell movement. This novel function may contribute to p53 anti-tumour activity.