N-methyladenosine (mA) modification of mRNA is emerging as an important regulator of gene expression that affects different developmental and biological processes, and altered mA homeostasis is linked to cancer. mA modification is catalysed by METTL3 and enriched in the 3' untranslated region of a large subset of mRNAs at sites close to the stop codon. METTL3 can promote translation but the mechanism and relevance of this process remain unknown. Here we show that METTL3 enhances translation only when tethered to reporter mRNA at sites close to the stop codon, supporting a mechanism of mRNA looping for ribosome recycling and translational control. Electron microscopy reveals the topology of individual polyribosomes with single METTL3 foci in close proximity to 5' cap-binding proteins. We identify a direct physical and functional interaction between METTL3 and the eukaryotic translation initiation factor 3 subunit h (eIF3h). METTL3 promotes translation of a large subset of oncogenic mRNAs-including bromodomain-containing protein 4-that is also mA-modified in human primary lung tumours. The METTL3-eIF3h interaction is required for enhanced translation, formation of densely packed polyribosomes and oncogenic transformation. METTL3 depletion inhibits tumorigenicity and sensitizes lung cancer cells to BRD4 inhibition. These findings uncover a mechanism of translation control that is based on mRNA looping and identify METTL3-eIF3h as a potential therapeutic target for patients with cancer.

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Wnts are important for various developmental and oncogenic processes. Here we show that Wnt signaling functions at synapses in hippocampal neurons. Tetanic stimulations induce N-methyl-Daspartate receptor-dependent synaptic Wnt3a release, nuclear ␤-catenin accumulations, and the activation of Wnt target genes. Suppression of Wnt signaling impairs long term potentiation. Conversely, activation of Wnt signaling facilitates long term potentiation. These findings suggest that Wnt signaling plays a critical role in regulating synaptic plasticity.Wnts are a family of secreted, lipid-modified signaling proteins that act as short range ligands to locally activate receptor-mediated signaling cascades. Wnt signaling is critical for a variety of developmental processes; its dysregulation is the causal factor for many diseases, especially cancer (1, 2). Wnt ligands bind to Frizzled receptors to activate intracellular signaling cascades, including the Wnt/␤-catenin, Wnt/Ca 2ϩ , and Wnt/planar cell polarity pathways (3). In the canonical Wnt/␤-catenin-signaling pathway, Frizzled receptors signal through Dishevelled to inhibit the kinase activity of glycogen synthase kinase 3␤ in a protein degradation complex containing Axin, ␤-catenin, and other proteins. When Wnt signaling is inactive, ␤-catenin is phosphorylated by glycogen synthase kinase 3␤ and thus rapidly degraded via the proteosome pathway; the activation of Wnt signaling stabilizes ␤-catenin by inhibiting glycogen synthase kinase 3␤. Stabilized ␤-catenin translocates to the nuclei and binds the TCF/LEF family of transcription factors to regulate the expression of Wnt target genes (1, 2). In addition, ␤-catenin is a component of the cadherin complex and plays an important role in regulating cell-cell adhesion (4). Among many Wnt-regulated developmental processes are neural patterning and differentiation (5), including hippocampal formation (6 -8), dendritic morphogenesis (9, 10), axon guidance (11-16), and synapse formation (11,17,18).Wnts are expressed in the brain (19 -21). Dysregulation of Wnt signaling has been suggested as an etiological cause for specific mental disorders. For example, Wnt signaling is up-regulated in schizophrenic brains (22)(23)(24). Several studies suggested an association of Frizzled-3 with schizophrenic susceptibility (25-27). On the other hand, downregulation of the Wnt/␤-catenin pathway is implicated in Alzheimer disease etiology (28 -34). A recent study indicated that cocaine exposures affect the expression of many Wnt signal-related genes (35). In addition, mutation of Dishevelled-1 in mice produces behavioral impairments (36), whereas mutation of a Drosophila Wnt receptor, Derailed (13), causes memory deficits (37, 38). Collectively, available data point to the role of Wnt signaling in the modulation of brain functions. However, the mechanism by which Wnt signaling regulates brain function is completely unknown.Here we report the synaptic localization of Wnt signaling proteins in mouse hippocampal neurons, the activity-induced Wnt3a rel...

Neurons differ in their responses to injury but the underlying mechanisms remain poorly understood. Using quantitative proteomics, we characterized the injury-triggered response from purified intact and axotomized retinal ganglion cells (RGCs). Subsequent informatics analyses revealed a network of injury-response signaling hubs. In addition to confirm known players, such as mTOR, this also identified new candidates, such as c-myc, NFkB and Huntingtin. Similar to mTOR, c-myc has been implicated as key regulators of anabolic metabolism and is down-regulated by axotomy. Forced expression of c-myc in RGCs, either before or after injury, promotes dramatic RGCs neuronal survival and axon regeneration after optic nerve injury. Finally, in contrast to RGCs, neither c-myc nor mTOR was down-regulated in injured peripheral sensory neurons. Our studies suggest that c-myc and other injury responsive pathways are critical to the intrinsic regenerative mechanisms and might represent a novel target for developing neural repair strategies in adults.

Local protein synthesis in neuronal dendrites is critical for synaptic plasticity. However, the signaling cascades that couple synaptic activation to dendritic protein synthesis remain elusive. The purpose of this study is to determine the role of glutamate receptors and the mammalian target of rapamycin (mTOR) signaling in regulating dendritic protein synthesis in live neurons. We first characterized the involvement of various subtypes of glutamate receptors and the mTOR kinase in regulating dendritic synthesis of a green fluorescent protein (GFP) reporter controlled by ␣CaMKII 5 and 3 untranslated regions in cultured hippocampal neurons. Specific antagonists of N-methyl-D-aspartic acid (NMDA), ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and metabotropic glutamate receptors abolished glutamate-induced dendritic GFP synthesis, whereas agonists of NMDA and metabotropic but not AMPA glutamate receptors activated GFP synthesis in dendrites. Inhibitions of the mTOR signaling, as well as its upstream activators, phosphatidylinositol 3-kinase and AKT, blocked NMDA receptor-dependent dendritic GFP synthesis. Conversely, activation of mTOR signaling stimulated dendritic GFP synthesis. In addition, we also found that inhibition of the mTOR kinase blocked dendritic synthesis of the endogenous ␣CaMKII and MAP2 proteins induced by tetanic stimulations in hippocampal slices. These results identify critical roles of NMDA receptors and the mTOR signaling pathway for control of synaptic activity-induced dendritic protein synthesis in hippocampal neurons.

Tauopathies, including Alzheimer's disease (AD), are associated with the aggregation of modified microtubule associated protein tau. This pathological state of tau is often referred to as “hyperphosphorylated”. Due to limitations in technology, an accurate quantitative description of this state is lacking. Here, a mass spectrometry-based assay, FLEXITau, is presented to measure phosphorylation stoichiometry and provide an unbiased quantitative view of the tau post-translational modification (PTM) landscape. The power of this assay is demonstrated by measuring the state of hyperphosphorylation from tau in a cellular model for AD pathology, mapping, and calculating site occupancies for over 20 phosphorylations. We further employ FLEXITau to define the tau PTM landscape present in AD post-mortem brain. As shown in this study, the application of this assay provides mechanistic understanding of tau pathology that could lead to novel therapeutics, and we envision its further use in prognostic and diagnostic approaches for tauopathies.

Osteosarcoma is the most common primary bone sarcoma that mostly occurs in young adults. The causes of osteosarcoma are heterogeneous and still not fully understood. Identification of novel, important oncogenic factors in osteosarcoma and development of better, effective therapeutic approaches are in urgent need for better treatment of osteosarcoma patients. In this study, we uncovered that the oncogene MYC is significantly upregulated in metastastic osteosarcoma samples. In addition, high MYC expression is associated with poor survival of osteosarcoma patients. Analysis of MYC targets in osteosarcoma revealed that most of the osteosarcoma super enhancer genes are bound by MYC. Treatment of osteosarcoma cells with super enhancer inhibitors THZ1 and JQ1 effectively suppresses the proliferation, migration, and invasion of osteosarcoma cells. Mechanistically, THZ1 treatment suppresses a large group of super enhancer containing MYC target genes including CDK6 and TGFB2. These findings revealed that the MYC-driven super enhancer signaling is crucial for the osteosarcoma tumorigenesis and targeting the MYC/super enhancer axis represents as a promising therapeutic strategy for treatment of osteosarcoma patients.

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