New Phosphospecific Antibody Reveals Isoform-Specific Phosphorylation of CPEB3 Protein

Cytoplasmic Polyadenylation Element Binding proteins (CPEBs) are a family of polyadenylation factors interacting with 3’UTRs of mRNA and thereby regulating gene expression. Various functions of CPEBs in development, synaptic plasticity, and cellular senescence have been reported. Four CPEB family members of partially overlapping functions have been described to date, each containing a distinct alternatively spliced region. This region is highly conserved between CPEBs-2-4 and contains a putative phosphorylation consensus, overlapping with the exon seven of CPEB3. We previously found CPEBs-2-4 splice isoforms containing exon seven to be predominantly present in neurons, and the isoform expression pattern to be cell type-specific. Here, focusing on the alternatively spliced region of CPEB3, we determined that putative neuronal isoforms of CPEB3 are phosphorylated. Using a new phosphospecific antibody directed to the phosphorylation consensus we found Protein Kinase A and Calcium/Calmodulin-dependent Protein Kinase II to robustly phosphorylate CPEB3 in vitro and in primary hippocampal neurons. Interestingly, status epilepticus induced by systemic kainate injection in mice led to specific upregulation of the CPEB3 isoforms containing exon seven. Extensive analysis of CPEB3 phosphorylation in vitro revealed two other phosphorylation sites. In addition, we found plethora of potential kinases that might be targeting the alternatively spliced kinase consensus site of CPEB3. As this site is highly conserved between the CPEB family members, we suggest the existence of a splicing-based regulatory mechanism of CPEB function, and describe a robust phosphospecific antibody to study it in future.     

CPEB4 Knockout Mice Exhibit Normal Hippocampus-Related Synaptic Plasticity and Memory

Regulated RNA translation is critical to provide proteins needed to maintain persistent modification of synaptic strength, which underlies the molecular basis of long-term memory (LTM). Cytoplasmic polyadenylation element-binding proteins (CPEBs) are sequence-specific RNA-binding proteins and regulate translation in various tissues. All four CPEBs in vertebrates are expressed in the brain, including the hippocampal neurons, suggesting their potential roles in translation-dependent plasticity and memory. Although CPEB1 and CPEB3 have been shown to control specific kinds of hippocampus-related LTM, the role of CPEB2 and CPEB4 in learning and memory remains elusive. Thus, we generated CPEB4 knockout (KO) mice and analyzed them using several behavioral tests. No difference was found in the anxiety level, motor coordination, hippocampus-dependent learning and memory between the KO mice and their wild-type (WT) littermates. Electrophysiological recordings of multiple forms of synaptic plasticity in the Schaffer collateral pathway-CA1 neurons also showed normal responses in the KO hippocampal slices. Morphological analyses revealed that the CPEB4-lacking pyramidal neurons possessed slightly elongated dendritic spines. Unlike its related family members, CPEB1 and CPEB3, CPEB4 seems to be dispensable for hippocampus-dependent plasticity, learning and memory.     

C. elegans CPB-3 interacts with DAZ-1 and functions in multiple steps of germline development

Cytoplasmic polyadenylation element-binding proteins (CPEBs) are well-conserved RNA-binding proteins, which regulate mRNA translation mainly through control of poly(A) elongation. Here, we show that CPB-3, one of the four CPEB homologs in C. elegans, positively regulates multiple aspects of oocyte production. CPB-3 protein was highly expressed in early meiotic regions of the hermaphrodite gonad. Worms deficient in cpb-3 were apparently impaired in germ cell proliferation and differentiation including sperm/oocyte switching and progression of female meiosis. We also show that cpb-3 is likely to promote the meiotic entry in parallel with gld-3, a component of one of the redundant but essential genetic pathways for the entry to and progression through meiosis. Taken together, CPEB appears to have a conserved role in the early phase of meiosis and in the sperm/oocyte specification, in addition to its reported function during meiotic progression.     

The Drosophila CPEB protein Orb2 has a novel expression pattern and is important for asymmetric cell division and nervous system function

Cytoplasmic polyadenylation element binding (CPEB) proteins bind mRNAs to regulate their localization and translation. While the first CPEBs discovered were germline specific, subsequent studies indicate that CPEBs also function in many somatic tissues including the nervous system. Drosophila has two CPEB family members. One of these, orb, plays a key role in the establishment of polarity axes in the developing egg and early embryo, but has no known somatic functions or expression outside of the germline. Here we characterize the other Drosophila CPEB, orb2. Unlike orb, orb2 mRNA and protein are found throughout development in many different somatic tissues. While orb2 mRNA and protein of maternal origin are distributed uniformly in early embryos, this pattern changes as development proceeds and by midembryogenesis the highest levels are found in the CNS and PNS. In the embryonic CNS, Orb2 appears to be concentrated in cell bodies and mostly absent from the longitudinal and commissural axon tracts. In contrast, in the adult brain, the protein is seen in axonal and dendritic terminals. Lethal effects are observed for both RNAi knockdowns and orb2 mutant alleles while surviving adults display locomotion and behavioral defects. We also show that orb2 funtions in asymmetric division of stem cells and precursor cells during the development of the embryonic nervous system and mesoderm.     

A reaction–diffusion mechanism influences cell lineage progression as a basis for formation, regeneration, and stability of intestinal crypts

Background

Translation efficiency of certain mRNAs can be regulated through a cytoplasmic polyadenylation process at the pre-initiation phase. A translational regulator controls the polyadenylation process and this regulation depends on its posttranslational modifications e.g., phosphorylation. The cytoplasmic polyadenylation binding protein (CPEB1) is one such translational regulator, which regulates the translation of some mRNAs by binding to the cytoplasmic polyadenylation element (CPE). The cytoplasmic polyadenylation process can be turned on or off by the phosphorylation or dephosphorylation state of CPEB1. A specific example could be the regulation of Calcium/Calmodulin-dependent protein kinase II (??CaMKII) translation through the phosphorylation/dephosphorylation cycle of CPEB1.

Result

Here, we show that CPEB1 mediated polyadenylation of ??CaMKII mRNA can result in a bistable switching mechanism. The switch for regulating the polyadenylation is based on a two state model of ??CaMKII and its interaction with CPEB1. Based on elementary biochemical kinetics a high dimensional system of non-linear ordinary differential equations can describe the dynamic characteristics of the polyadenylation loop. Here, we simplified this high-dimensional system into approximate lower dimension system that can provide the understanding of dynamics and fixed points of original system. These simplified equations can be used to develop analytical bifurcation diagrams without the use of complex numerical tracking algorithm, and can further give us intuition about the parameter dependence of bistability in this system.

Conclusion

This study provides a systematic method to simplify, approximate and analyze a translation/activation based positive feedback loop. This work shows how to extract low dimensional systems that can be used to obtain analytical solutions for the fixed points of the system and to describe the dynamics of the system. The methods used here have general applicability to the formulation and analysis of many molecular networks.     

Novel RNA recognition motif domain in the cytoplasmic polyadenylation element binding protein 3

The family of cytoplasmic polyadenylation element binding proteins CPEB1, CPEB2, CPEB3, and CPEB4 binds to the 3′‐untranslated region (3′‐UTR) of mRNA, and plays significant roles in mRNA metabolism and translation regulation. They have a common domain organization, involving two consecutive RNA recognition motif (RRM) domains followed by a zinc finger domain in the C‐terminal region. We solved the solution structure of the first RRM domain (RRM1) of human CPEB3, which revealed that CPEB3 RRM1 exhibits structural features distinct from those of the canonical RRM domain. Our structural data provide important information about the RNA binding ability of CPEB3 RRM1. Proteins 2014; 82:2879–2886. © 2014 Wiley Periodicals, Inc.     

The clam 3' UTR masking element-binding protein p82 is a member of the CPEB family.

During early development gene expression is controlled principally at the translational level. Oocytes of the surf clam Spisula solidissima contain large stockpiles of maternal mRNAs that are translationally dormant or masked until meiotic maturation. Activation of the oocyte by fertilization leads to translational activation of the abundant cyclin and ribonucleotide reductase mRNAs at a time when they undergo cytoplasmic polyadenylation. In vitro unmasking assays have defined U-rich regions located approximately centrally in the 3' UTRs of these mRNAs as translational masking elements. A clam oocyte protein of 82 kDa, p82, which selectively binds the masking elements, has been proposed to act as a translational repressor. Importantly, mRNA-specific unmasking in vitro occurs in the absence of poly(A) extension. Here we show that clam p82 is related to Xenopus CPEB, an RNA-binding protein that interacts with the U-rich cytoplasmic polyadenylation elements (CPEs) of maternal mRNAs and promotes their polyadenylation. Cloned clam p82/CPEB shows extensive homology to Xenopus CPEB and related polypeptides from mouse, goldfish, Drosophila and Caenorhabditis elegans, particularly in their RNA-binding C-terminal halves. Two short N-terminal islands of sequence, of unknown function, are common to vertebrate CPEBs and clam p82. p82 undergoes rapid phosphorylation either directly or indirectly by cdc2 kinase after fertilization in meiotically maturing clam oocytes, prior to its degradation during the first cell cleavage. Phosphorylation precedes and, according to inhibitor studies, may be required for translational activation of maternal mRNA. These data suggest that clam p82 may be a functional homolog of Xenopus CPEB.     

CPEB3 Deficiency Elevates TRPV1 Expression in Dorsal Root Ganglia Neurons to Potentiate Thermosensation

Cytoplasmic polyadenylation element binding protein 3 (CPEB3) is a sequence-specific RNA-binding protein that downregulates translation of multiple plasticity-related proteins (PRPs) at the glutamatergic synapses. Activity-induced synthesis of PRPs maintains long-lasting synaptic changes that are critical for memory consolidation and chronic pain manifestation. CPEB3-knockout (KO) mice show aberrant hippocampus-related plasticity and memory, so we investigated whether CPEB3 might have a role in nociception-associated plasticity. CPEB3 is widely expressed in the brain and peripheral afferent sensory neurons. CPEB3-KO mice with normal mechanosensation showed hypersensitivity to noxious heat. In the complete Freund''s adjuvant (CFA)-induced inflammatory pain model, CPEB3-KO animals showed normal thermal hyperalgesia and transiently enhanced mechanical hyperalgesia. Translation of transient receptor potential vanilloid 1 (TRPV1) RNA was suppressed by CPEB3 in dorsal root ganglia (DRG), whereas CFA-induced inflammation reversed this inhibition. Moreover, CPEB3/TRPV1 double-KO mice behaved like TRPV1-KO mice, with severely impaired thermosensation and thermal hyperalgesia. An enhanced thermal response was recapitulated in non-inflamed but not inflamed conditional-KO mice, with cpeb3 gene ablated mostly but not completely, in small-diameter nociceptive DRG neurons. CPEB3-regulated translation of TRPV1 RNA may play a role in fine-tuning thermal sensitivity of nociceptors.     

Muscarinic signaling influences the patterning and phenotype of cholinergic amacrine cells in the developing chick retina

Background  

Many studies in the vertebrate retina have characterized the differentiation of amacrine cells as a homogenous class of neurons, but little is known about the genes and factors that regulate the development of distinct types of amacrine cells. Accordingly, the purpose of this study was to characterize the development of the cholinergic amacrine cells and identify factors that influence their development. Cholinergic amacrine cells in the embryonic chick retina were identified by using antibodies to choline acetyltransferase (ChAT).     

Proteome profiling of embryo chick retina

Background  

Little is known regarding the molecular pathways that underlie the process of retinal development. The purpose of this study was to identify proteins which may be involved in development of retina. We used a proteomics-based approach to identify proteins that are up- or down-regulated during the development of the embryo chick retina.     

CPEB3 deficiency in mice affect ovarian follicle development and causes premature ovarian insufficiency

Premature ovarian insufficiency (POI) is a heterogeneous and multifactorial disorder. In recent years, there has been an increasing interest in research on the pathogenesis and treatment of POI, owing to the implementation of the second-child policy in China. Cytoplasmic polyadenylation element-binding protein 3 (CPEB3) is an RNA-binding protein that can bind to specific RNA sequences. CPEB3 can bind to and affect the expression, cellular location, and stability of target RNAs. Cpeb3 is highly expressed in the ovary; however, its functions remain unknown. In this study, Cpeb3-mutant mice were used to characterize the physiological functions of CPEB3. Cpeb3-mutant female mice manifested signs of gradual loss of ovarian follicles, ovarian follicle development arrest, increased follicle atresia, and subfertility with a phenotype analogous to POI in women. Further analysis showed that granulosa cell proliferation was inhibited and apoptosis was markedly increased in Cpeb3-mutant ovaries. In addition, the expression of Gdf9, a potential target of CPEB3, was decreased in Cpeb3-mutant ovaries and oocytes. Altogether, these results reveal that CPEB3 is essential for ovarian follicle development and female fertility as it regulates the expression of Gdf9 in oocytes, disruption of which leads to impaired ovarian follicle development and POI.Subject terms: RNA-binding proteins, Infertility