CPEB1 regulates the inflammatory immune response, phagocytosis, and alternative polyadenylation in microglia.

Microglia are myeloid cells of the central nervous system that perform tasks essential for brain development, neural circuit homeostasis, and neural disease. Microglia react to inflammatory stimuli by upregulating inflammatory signaling through several different immune cell receptors such as the Toll-like receptor 4 (TLR4), which signals to several downstream effectors including transforming growth factor beta-activated kinase 1 (TAK1). Here, we show that TAK1 levels are regulated by CPEB1, a sequence-specific RNA binding protein that controls translation as well as RNA splicing and alternative poly(A) site selection in microglia. Lipopolysaccharide (LPS) binds the TLR4 receptor, which in CPEB1-deficient mice leads to elevated expression of ionized calcium binding adaptor molecule 1 (Iba1), a microglial protein that increases with inflammation, and increased levels of the cytokine IL6. This LPS-induced IL6 response is blocked by inhibitors of JNK, p38, ERK, NFκB, and TAK1. In contrast, phagocytosis, which is elevated in CPEB1-deficient microglia, is unaffected by LPS treatment or ERK inhibition, but is blocked by TAK1 inhibition. These data indicate that CPEB1 regulates microglial inflammatory responses and phagocytosis. RNA-seq indicates that these changes in inflammation and phagocytosis are accompanied by changes in RNA levels, splicing, and alternative poly(A) site selection. Thus, CPEB1 regulation of RNA expression plays a role in microglial function.

EGFP insertional mutagenesis reveals multiple FXR2P fibrillar states with differing ribosome association in neurons.

RNA-binding proteins (RBPs) function in higher-order assemblages such as RNA granules to regulate RNA localization and translation. The Fragile X homolog FXR2P is an RBP essential for formation of neuronal Fragile X granules that associate with axonal mRNA and ribosomes in the intact brain. However, the FXR2P domains important for assemblage formation in a cellular system are unknown. Here we used an EGFP insertional mutagenesis approach to probe for FXR2P intrinsic features that influence its structural states. We tested 18 different in-frame FXR2PEGFP fusions in neurons and found that the majority did not impact assemblage formation. However, EGFP insertion within a 23 amino acid region of the low complexity (LC) domain induced FXR2PEGFP assembly into two distinct fibril states that were observed in isolation or in highly-ordered bundles. FXR2PEGFP fibrils exhibited different developmental timelines, ultrastructures and ribosome associations. Formation of both fibril types was dependent on an intact RNA-binding domain. These results suggest that restricted regions of the LC domain, together with the RNA-binding domain, may be important for FXR2P structural state organization in neurons.

Essential role for non-canonical poly(A) polymerase GLD4 in cytoplasmic polyadenylation and carbohydrate metabolism.

Regulation of gene expression at the level of cytoplasmic polyadenylation is important for many biological phenomena including cell cycle progression, mitochondrial respiration, and learning and memory. GLD4 is one of the non-canonical poly(A) polymerases that regulates cytoplasmic polyadenylation-induced translation, but its target mRNAs and role in cellular physiology is not well known. To assess the full panoply of mRNAs whose polyadenylation is controlled by GLD4, we performed an unbiased whole genome-wide screen using poy(U) chromatography and thermal elution. We identified hundreds of mRNAs regulated by GLD4, several of which are involved in carbohydrate metabolism including GLUT1, a major glucose transporter. Depletion of GLD4 not only reduced GLUT1 poly(A) tail length, but also GLUT1 protein. GLD4-mediated translational control of GLUT1 mRNA is dependent of an RNA binding protein, CPEB1, and its binding elements in the 3΄ UTR. Through regulating GLUT1 level, GLD4 affects glucose uptake into cells and lactate levels. Moreover, GLD4 depletion impairs glucose deprivation-induced GLUT1 up-regulation. In addition, we found that GLD4 affects glucose-dependent cellular phenotypes such as migration and invasion in glioblastoma cells. Our observations delineate a novel post-transcriptional regulatory network involving carbohydrate metabolism and glucose homeostasis mediated by GLD4.

Gld2-catalyzed 3' monoadenylation of miRNAs in the hippocampus has no detectable effect on their stability or on animal behavior.

Gld2, a noncanonical cytoplasmic poly(A) polymerase, interacts with the RNA binding protein CPEB1 to mediate polyadenylation-induced translation in dendrites of cultured hippocampal neurons. Depletion of Gld2 from the hippocampus leads to a deficit in long-term potentiation evoked by theta burst stimulation. At least in mouse liver and human primary fibroblasts, Gld2 also 3' monoadenylates and thereby stabilizes specific miRNAs, which enhance mRNA translational silencing and eventual destruction. These results suggest that Gld2 would be likely to monoadenylate and stabilize miRNAs in the hippocampus, which would produce measurable changes in animal behavior. We now report that using Gld2 knockout mice, there are detectable alterations in specific miRNA monoadenylation in the hippocampus when compared to wild type, but that these modifications produce no detectable effect on miRNA stability. Moreover, we surprisingly find no overt change in animal behavior when comparing Gld2 knockout to wild-type mice. These data indicate that miRNA monoadenylation-mediated stability is cell type-specific and that monoadenylation has no measurable effect on higher cognitive function.

The Centrosome Undergoes Plk1-Independent Interphase Maturation during Inflammation and Mediates Cytokine Release.

Cytokine production is a necessary event in the immune response during inflammation and is associated with mortality during sepsis, autoimmune disorders, cancer, and diabetes. Stress-activated MAP kinase signaling cascades that mediate cytokine synthesis are well established. However, the downstream fate of cytokines before they are secreted remains elusive. We report that pro-inflammatory stimuli lead to recruitment of pericentriolar material, specifically pericentrin and γ-tubulin, to the centrosome. This is accompanied by enhanced microtubule nucleation and enrichment of the recycling endosome component FIP3, all of which are hallmarks of centrosome maturation during mitosis. Intriguingly, centrosome maturation occurs during interphase in an MLK-dependent manner, independent of the classic mitotic kinase, Plk1. Centrosome disruption by chemical prevention of centriole assembly or genetic ablation of pericentrin attenuated interleukin-6, interleukin-10, and MCP1 secretion, suggesting that the centrosome is critical for cytokine production. Our results reveal a function of the centrosome in innate immunity.

Centrosome-intrinsic mechanisms modulate centrosome integrity during fever.

The centrosome is critical for cell division, ciliogenesis, membrane trafficking, and immunological synapse function. The immunological synapse is part of the immune response, which is often accompanied by fever/heat stress (HS). Here we provide evidence that HS causes deconstruction of all centrosome substructures primarily through degradation by centrosome-associated proteasomes. This renders the centrosome nonfunctional. Heat-activated degradation is centrosome selective, as other nonmembranous organelles (midbody, kinetochore) and membrane-bounded organelles (mitochondria) remain largely intact. Heat-induced centrosome inactivation was rescued by targeting Hsp70 to the centrosome. In contrast, Hsp70 excluded from the centrosome via targeting to membranes failed to rescue, as did chaperone inactivation. This indicates that there is a balance between degradation and chaperone rescue at the centrosome after HS. This novel mechanism of centrosome regulation during fever contributes to immunological synapse formation. Heat-induced centrosome inactivation is a physiologically relevant event, as centrosomes in leukocytes of febrile patients are disrupted.

CPEB regulation of TAK1 synthesis mediates cytokine production and the inflammatory immune response.

The cytoplasmic-element-binding (CPEB) protein is a sequence-specific RNA-binding protein that regulates cytoplasmic polyadenylation-induced translation. In mouse embryo fibroblasts (MEFs) lacking CPEB, many mRNAs encoding proteins involved in inflammation are misregulated. Correlated with this aberrant translation in MEFs, a macrophage cell line depleted of CPEB and treated with lipopolysaccharide (LPS) to stimulate the inflammatory immune response expresses high levels of interleukin-6 (IL-6), which is due to prolonged nuclear retention of NF-κB. Two proteins involved in NF-κB nuclear localization and IL-6 expression, IκBα and transforming growth factor beta-activated kinase 1 (TAK1), are present at excessively low and high steady-state levels, respectively, in LPS-treated CPEB-depleted macrophages. However, only TAK1 has an altered synthesis rate that is CPEB dependent and CPEB/TAK1 double depletion alleviates high IL-6 production. Peritoneal macrophages isolated from CPEB knockout (KO) mice treated with LPS in vitro also have prolonged NF-κB nuclear retention and produce high IL-6 levels. LPS-injected CPEB KO mice secrete prodigious amounts of IL-6 and other proinflammatory cytokines and exhibit hypersensitivity to endotoxic shock; these effects are mitigated when the animals are also injected with (5Z)-7-oxozeaenol, a potent and specific inhibitor of TAK1. These data show that CPEB control of TAK1 mRNA translation mediates the inflammatory immune response.

Cytoplasmic polyadenylation element binding proteins in development, health, and disease.

The cytoplasmic polyadenylation element binding (CPEB) proteins are sequence-specific mRNA binding proteins that control translation in development, health, and disease. CPEB1, the founding member of this family, has become an important model for illustrating general principles of translational control by cytoplasmic polyadenylation in gametogenesis, cancer etiology, synaptic plasticity, learning, and memory. Although the biological functions of the other members of this protein family in vertebrates are just beginning to emerge, it is already evident that they, too, mediate important processes, such as cancer etiology and higher cognitive function. In Drosophila, the CPEB proteins Orb and Orb2 play key roles in oogenesis and in neuronal function, as do related proteins in Caenorhabditis elegans and Aplysia. We review the biochemical features of the CPEB proteins, discuss their activities in several biological systems, and illustrate how understanding CPEB activity in model organisms has an important impact on neurological disease.

Genetic and acute CPEB1 depletion ameliorate fragile X pathophysiology.

Fragile X syndrome (FXS), the most common cause of inherited mental retardation and autism, is caused by transcriptional silencing of FMR1, which encodes the translational repressor fragile X mental retardation protein (FMRP). FMRP and cytoplasmic polyadenylation element-binding protein (CPEB), an activator of translation, are present in neuronal dendrites, are predicted to bind many of the same mRNAs and may mediate a translational homeostasis that, when imbalanced, results in FXS. Consistent with this possibility, Fmr1(-/y); Cpeb1(-/-) double-knockout mice displayed amelioration of biochemical, morphological, electrophysiological and behavioral phenotypes associated with FXS. Acute depletion of CPEB1 in the hippocampus of adult Fmr1(-/y) mice rescued working memory deficits, demonstrating reversal of this FXS phenotype. Finally, we find that FMRP and CPEB1 balance translation at the level of polypeptide elongation. Our results suggest that disruption of translational homeostasis is causal for FXS and that the maintenance of this homeostasis by FMRP and CPEB1 is necessary for normal neurologic function.

Cytoplasmic polyadenylation element binding protein deficiency stimulates PTEN and Stat3 mRNA translation and induces hepatic insulin resistance.

The cytoplasmic polyadenylation element binding protein CPEB1 (CPEB) regulates germ cell development, synaptic plasticity, and cellular senescence. A microarray analysis of mRNAs regulated by CPEB unexpectedly showed that several encoded proteins are involved in insulin signaling. An investigation of Cpeb1 knockout mice revealed that the expression of two particular negative regulators of insulin action, PTEN and Stat3, were aberrantly increased. Insulin signaling to Akt was attenuated in livers of CPEB-deficient mice, suggesting that they might be defective in regulating glucose homeostasis. Indeed, when the Cpeb1 knockout mice were fed a high-fat diet, their livers became insulin-resistant. Analysis of HepG2 cells, a human liver cell line, depleted of CPEB demonstrated that this protein directly regulates the translation of PTEN and Stat3 mRNAs. Our results show that CPEB regulated translation is a key process involved in insulin signaling.

Control of cellular senescence by CPEB.

Cytoplasmic polyadenylation element-binding protein (CPEB) is a sequence-specific RNA-binding protein that promotes polyadenylation-induced translation. While a CPEB knockout (KO) mouse is sterile but overtly normal, embryo fibroblasts derived from this mouse (MEFs) do not enter senescence in culture as do wild-type MEFs, but instead are immortal. Exogenous CPEB restores senescence in the KO MEFs and also induces precocious senescence in wild-type MEFs. CPEB cannot stimulate senescence in MEFs lacking the tumor suppressors p53, p19ARF, or p16(INK4A); however, the mRNAs encoding these proteins are unlikely targets of CPEB since their expression is the same in wild-type and KO MEFs. Conversely, Ras cannot induce senescence in MEFs lacking CPEB, suggesting that it may lie upstream of CPEB. One target of CPEB regulation is myc mRNA, whose unregulated translation in the KO MEFs may cause them to bypass senescence. Thus, CPEB appears to act as a translational repressor protein to control myc translation and resulting cellular senescence.

A novel phosphatidylinositol(3,4,5)P3 pathway in fission yeast.

The mammalian tumor suppressor, phosphatase and tensin homologue deleted on chromosome 10 (PTEN), inhibits cell growth and survival by dephosphorylating phosphatidylinositol-(3,4,5)-trisphosphate (PI[3,4,5]P3). We have found a homologue of PTEN in the fission yeast, Schizosaccharomyces pombe (ptn1). This was an unexpected finding because yeast (S. pombe and Saccharomyces cerevisiae) lack the class I phosphoinositide 3-kinases that generate PI(3,4,5)P3 in higher eukaryotes. Indeed, PI(3,4,5)P3 has not been detected in yeast. Surprisingly, upon deletion of ptn1 in S. pombe, PI(3,4,5)P3 became detectable at levels comparable to those in mammalian cells, indicating that a pathway exists for synthesis of this lipid and that the S. pombe ptn1, like mammalian PTEN, suppresses PI(3,4,5)P3 levels. By examining various mutants, we show that synthesis of PI(3,4,5)P3 in S. pombe requires the class III phosphoinositide 3-kinase, vps34p, and the phosphatidylinositol-4-phosphate 5-kinase, its3p, but does not require the phosphatidylinositol-3-phosphate 5-kinase, fab1p. These studies suggest that a pathway for PI(3,4,5)P3 synthesis downstream of a class III phosphoinositide 3-kinase evolved before the appearance of class I phosphoinositide 3-kinases.

In vitro FRAP reveals the ATP-dependent nuclear mobilization of the exon junction complex protein SRm160.

We present a new in vitro system for characterizing the binding and mobility of enhanced green fluorescent protein (EGFP)-labeled nuclear proteins by fluorescence recovery after photobleaching in digitonin-permeabilized cells. This assay reveals that SRm160, a splicing coactivator and component of the exon junction complex (EJC) involved in RNA export, has an adenosine triphosphate (ATP)-dependent mobility. Endogenous SRm160, lacking the EGFP moiety, could also be released from sites at splicing speckled domains by an ATP-dependent mechanism. A second EJC protein, RNPS1, also has an ATP-dependent mobility, but SRm300, a protein that binds to SRm160 and participates with it in RNA splicing, remains immobile after ATP supplementation. This finding suggests that SRm160-containing RNA export, but not splicing, complexes have an ATP-dependent mobility. We propose that RNA export complexes have an ATP-regulated mechanism for release from binding sites at splicing speckled domains. In vitro fluorescence recovery after photobleaching is a powerful tool for identifying cofactors required for nuclear binding and mobility.

The mRNA-binding protein YB-1 (p50) prevents association of the eukaryotic initiation factor eIF4G with mRNA and inhibits protein synthesis at the initiation stage.

The cytoplasmic messenger ribonucleoprotein particles of mammalian somatic cells contain the protein YB-1, also called p50, as a major core component. YB-1 is multifunctional and involved in regulation of mRNA transcription and translation. Our previous studies demonstrated that YB-1 stimulates initiation of translation in vitro at a low YB-1/mRNA ratio, whereas an increase of YB-1 bound to mRNA resulted in inhibition of protein synthesis in vitro and in vivo. Here we show that YB-1-mediated translation inhibition in a rabbit reticulocyte cell-free system is followed by a decay of polysomes, which is not a result of mRNA degradation or its functional inactivation. The inhibition does not change the ribosome transit time, and therefore, it affects neither elongation nor termination of polypeptide chains and only occurs at the stage of initiation. YB-1 induces accumulation of mRNA in the form of free messenger ribonucleoprotein particles, i.e. it blocks mRNA association with the small ribosomal subunit. The accumulation is accompanied by eukaryotic initiation factor eIF4G dissociation from mRNA. The C-terminal domain of YB-1 is responsible for inhibition of translation as well as the disruption of mRNA interaction with eIF4G.